Quant ita tiv e appr oac h for dete ct ion of RNA and prot eins by s and w ic h hy bridiza tion te chno logy vorgelegt v on M Sc . Ekaterina O smekhina ORC ID: 0000 - 0002 - 5756 - 9877 von d er Fakul tät III - Prozesswissenscha ften der Technischen U niversi tät B erlin zur Erla ng ung des a k ade mis chen G ra des Doktor de r Ingenieurwissenschaften – Dr. - Ing – g enehmigte Dissertation Promoti onsausschu ss: Vorsitzender: Prof. D r. Roland Laust e r Gutac hter: Prof. Dr. Pete r N euba uer Gutac hter: Prof. Dr. Loren z Adrian Gutac hter: Prof . Dr . Le if - Alexander G arbe Tag der wiss enschaftlichen Aussprach e: 16. N ovember 2018 Berlin 2019 A b stra ct The detec tion and q uanti f ication of prot eins and nu cleic acids is an import ant procedure across a wi de variety o f biological disciplines and has forever changed and improved our ability to accurat ely determine u nique trait s in sa m ple consistency . In this thesis, we show t he applicability of a sandwich hybridization technolog y f or th e detection and quanti f ication of ( i ) collagen prolyl -4- hydroxyl ase expression , ( ii ) Bacillus subt ilis air borne spores and (ii) Salmonella in food samples. F or collagen prolyl -4 - hydroxy lase det ecti on, we dev eloped a sandwich ELISA utilizing antibodies for tw o different subunits of the protein, optimized the procedure and the antib ody concent rations using ex per imental design approach , and applied the met hod to monitor the recombinant protein concentration in crude cell extracts during its production in E. coli . To detect Bacillus subt ilis spores from the air, w e est ablished a procedure that includes spore ac t ivation, germination, and enrichment cul t ivation, cell disruption, and RNA analys is us ing a sa ndwich hybridization assay (SHA). Using this process w e were able to detect less t han 10 s por es in a sing le sample a fter 5 hours of enrich m ent cultiv ation. To analyze food samples f or Salmonella contamina tion , we developed an improved enrichment cultivation procedure toge ther w ith a SHA detection method. Salmonella 23S r RNA wa s selected as a target for the SHA. The SHA showed high selectivity toward S. Typhimurium , even in the presence o f high concentration o f background fl ora . Moreov er , using the modif ied media, we achi eved a significant improvement in the rec overy o f heat injur ed Salmonella cells. In this t hesis we show t hat sandwich hy br idization technology is a f a st and convenient tool f or the detect ion of a w ide range of biomolecules i n a variety of samples, demonstrating its potential and illustrating the possibilities f or f urther dev elopment. Zusa mmenf a ssung Der Nachweis und di e Q uantifizierung von Pro teinen und Nukleinsäuren ist ein w ichti ges Verfahr en in einer Vielzahl von bi ologischen Disziplinen und ha t unsere Fähigkeit , einzigart ige M erk male in de r Probenkonsistenz g enau zu bes t immen , für im m er verändert und verbessert. In dieser Arbeit w ird das Potential der Sandwi ch - Hybridisierungst ec hnologie für die Quantif izierung v on Zielmolekülen in verschiedenen Anw endung en a ufgezeigt: (1) die funkt ionelle Ex pr ession von Kollagen - P r ol yl -4- Hydroxylase, (2) für den q uantitativen Nachweis von Bacillus - Sporen und (3) für die Detektion von Sal monellen in Lebensmitt elproben. Für den Nachweis von Kol lagen - Pr o lyl -4- Hy dro xylase w u r de ein S andwich - ELISA unte r Verwendung von Antikörpern für zw ei verschiedenen Untereinheiten des Enz yms entwickelt und mit H ilfe des stat istisch basierten E xperimentellen Designs (DoE) opt imiert. Dieser Assay w urde dann zur Bestimmung der he t erologen Expression d er Kollagen - Pr o lyl -4- Hydroxylase in einem Escherichia coli Fed - ba tch Prozess an g ewandt. Z ur Detektion von B acillus -S poren in de r Luft, wurde am Beispiel von Bacillu s sub til is ein Verf ahren etabliert, das Sporenakt ivierung, Keimung und Anreicherungskultivi erung , Zellau f schluss und R NA - Analyse unter Verw endung eines Sandw ich - Hybrid is i erung s - Assays (SHA) umfasst. Mit diesem Ve rf ahren konnten nach fünf Stunden Anreicher ungskultivierung in einer Probe weni ger als 10 Sporen nachgewi esen werden. Schließlich w ur de zur Detektion von Sal monellenk ontaminationen in Nahrungsmittelproben ein v erbessertes Anreicherungsanbauverfahren zusammen mit ei ner Sandwich - Hy bridisierung als Nachw eism ethode entw ick elt . Der au f der quantitativen Detektion von 23S rRN A basier ende Assay zeigte eine hohe Selektivität gegenüber S. Typhimurium, sogar in Gegenwart einer hohen Konz ent rat ion der Hintergrundflora. Darüber hinaus wurde bei der Anreicherung durch die V erwendung modifiziert er M edien eine signifikant e Erhöhun g der Ausbeute an Sal monella - RN A aus vorher hi tzegeschädigten P roben erhalten und damit eine wesentliche Erhöhung der Sensitivität. Zusammen f assend konnt e in dieser Arbeit das Potential der Sandw ic h- Hybridisierung stechnologie als ei ne einfach z u etablierende und sehr s ensitive Methode zum quantitat iven Nachw eis von Biomolekülen f ür untersc hiedliche Anw endung sbereiche in der mi k robiellen D iagnost ik aufgez eig t werden, was trotz neuer alternativer Entwicklung en auf dem Gebiet, das Potential der Methode dokumentiert. Table of C ont ents TITLE ABSTRAC T ZUSAMM EN FA SSUNG TABLE OF CON TENTS ABBREVI ATIONS ......................................................................................................................................................... 1 AC KNOW LEDGM E NTS ................................................................................................................................................. 4 LIST OF PUBLIC ATIO NS ................................................................................................................................................ 5 GENERAL INTR ODU CTION .................................................................................................................................. 6 INRODUCTION ................................................................................................................................................. 6 PROTEIN - BASE D DE TE CTION ............................................................................................................................ 6 1.2.1. ENZYM E - LINK ED IMMU NOSORBENT ASSAY S (ELISAs ) ................................................................................ 7 NUCLE IC A CID - BASE D DE TEC TI ON ................................................................................................................. 10 1.3.1. BLO TTIN G ME THO DS ................................................................................................................................ 10 1.3.2. MICROARRAYS .......................................................................................................................................... 10 1.3.3. RNA SEQ UENCING (RNA - seq) .................................................................................................................... 11 1.3.4. QUAN TI TA TIVE PCR (q PCR ) ....................................................................................................................... 11 1.3.5. SANDW ICH H YBRI DIZAT ION ASSAY S (SHA) ............................................................................................... 12 AIMS OF THE PRESENT ST UDY ........................................................................................................................ 14 SAND WIC H ELIS A FOR Q UAN TITATIV E DET ECTIO N OF HUMAN COLLAGE N PR OLYL 4 - H YDROXY LASE ............... 16 INTRODUCTION ............................................................................................................................................. 16 2.1.1. COLLA GE N PR OL YL 4 - H YDR OX YL ASE S ....................................................................................................... 16 2.1.2. DETE CTI ON OF CP4 H ................................................................................................................................ . 18 MATERIALS AND M E THO DS ........................................................................................................................... 18 2.2.1. STR AIN AND CULTIVAT I ON C ONDIT IONS .................................................................................................. 18 2.2.2. ANAL YSIS O F RECOMB INANT C P4H ........................................................................................................... 19 2.2.3. ANTIBODIES............................................................................................................................................... 20 2.2.4. SANDWICH ELISA (OPTIMIZE D PROTOCOL) .............................................................................................. 20 2.2.5. EXPER IMENT AL D ESIGN A ND STAT ISTIC AL A NALYSIS ............................................................................... 21 RESUL TS ......................................................................................................................................................... 22 2.3.1. METH OD D EVEL OPM ENT .......................................................................................................................... 22 2.3.2. M ab - α ATT A CHMENT TO MAGNETI C BEA DS ............................................................................................ 23 2.3.3. CHO OSI NG THE OP TIMA L pa b - β ............................................................................................................... 24 2.3.4. OPTIM IZ ATI ON OF THE ME TH OD .............................................................................................................. 26 2.3.5. OPTIMI Z ATI ON OF TH E AN TIB ODY CON CE NTRA TION S ............................................................................. 27 2.3.6. SENSITI VIT Y AND R ANGE ........................................................................................................................... 33 2.3.7. ANAL YSIS O F THE RECO MBI NANT CP 4H EXPRESSI ON IN ESC HERICHIA COLI ............................................ 34 2.3.8. OPTIMI Z ATI ON OF A FED - BATCH FER MENT ATIO N PROC ESS FOR C P4H P RODUC TION I N ESC HERIC HIA COLI … …… ………… …… ………… … …………… …… ………… …… ………… ……… ………… ………… …… …… …………… ………… …… ………… .. 36 DISCUSSI ON ................................................................................................................................................... 41 RNA B ASED D ETECT ION OF B ACILL US SPOR ES .................................................................................................. 43 INTRODUCTION ............................................................................................................................................. 43 3.1.1. BACILLUS ANTHRACIS ................................................................................................................................ 43 3.1.2. BACILLUS SUBTILIS .................................................................................................................................... 46 3.1.3. EXIST ING MET HODS FO R SPOR E DETEC TION ............................................................................................ 49 3.1.4. RNA B ASED DETEC TION OF BAC ILLU S SPOR ES .......................................................................................... 53 MATERIALS AND M E THO DS ........................................................................................................................... 54 3.2.1. SPORE PRE PARATI ON ................................................................................................................................ 54 3.2.2. A CTIV ATI ON AN D GR OW TH C ON DITI ON S ................................................................................................ . 54 3.2.3. SAMPLE PRE PARATION ............................................................................................................................. 55 3.2.4. GENER ATI ON OF T HE IN VIT RO 16 S rRNA TRANSC RIPT ............................................................................ 55 3.2.5. OLIG ONUCL E OTI DE PROBE S ...................................................................................................................... 57 3.2.6. SANDWICH HYBRIDIZA TION ASSAY ........................................................................................................... 57 RESUL TS ......................................................................................................................................................... 59 3.3.1. RNA B ASED DETEC TION OF BAC ILLU S SPOR ES .......................................................................................... 59 3.3.2. ACT I VAT I ON AND CULT I VAT I ON OF BA CILLUS SUB TILIS SPORES .............................................................. 60 3.3.3. DEVEL OPM ENT OF SHA FOR B. SU BT ILIS 1 6S r RNA .................................................................................. 60 3.3.4. DET ECT ION OF B. SUB TILIS SPO RES .......................................................................................................... 64 3.3.5. ESTIM ATION OF 16S rR NA MOLECU LE LEVEL S IN B. SU BTILI S CELLS ........................................................ 65 DISCUSSI ON ................................................................................................................................................... 66 RNA B ASED D ETECT ION OF S ALMONE LLA ......................................................................................................... 69 INTRODUCTION ............................................................................................................................................. 69 4.1.1. DISEA SE ..................................................................................................................................................... 69 4.1.2. ANTI BIO TI C RESISTANC E ........................................................................................................................... 70 4.1.3. DETE CTI ON ................................................................................................................................................ 73 MATERIALS AND M E THO DS ........................................................................................................................... 77 4.2.1. STR AINS ..................................................................................................................................................... 77 4.2.2. CULTIVATION MEDIA AND CO NDITIONS ................................................................................................... 77 4.2.3. PREPA RATION O F HEAT - INJU RED CELL S ................................................................................................... 78 4.2.4. MEAT SAMPLES ......................................................................................................................................... 78 4.2.5. GROWTH C URVES ..................................................................................................................................... 78 4.2.6. CELL LYSIS .................................................................................................................................................. 79 4.2.7. OLIG ONUCL EOTIDE PRO BES FOR SHA ....................................................................................................... 79 4.2.8. GENER ATI ON OF T HE IN VIT RO 23 S rRNA TRANSC RIPT ............................................................................ 79 4.2.9. ANAL YSIS O F THE 23 S rRNA AMO UNTS .................................................................................................... 80 RESUL TS ......................................................................................................................................................... 82 4.3.1. DEVEL OPM ENT OF SHA FOR S ALMONELLA T YPHIM URIUM 23rRNA ........................................................ 82 4.3.2. DET ECT ION OF SALMONEL LA T YP HIMURIU M IN MEA T SA MPLES USING SHA ......................................... 85 DISCUSSI ON ................................................................................................................................................... 87 SUMM ARY O F RES ULT S A N D OUTLOO K ............................................................................................................ 89 SANDW ICH H YBR I DIZ ATI ON FOR PR O TEI N DE TE CTI ON ................................................................................. 89 SANDW ICH H YBR I DIZ ATI ON FOR SPORE DE TE CTI ON ..................................................................................... 90 SANDW ICH H YBR I DIZ ATI ON FOR FOOD P ATHO GE N DETE C TI ON ................................................................... 90 ADVANTAGES OF SAND WICH H YBRIDIZATION TECHNOLOGY ....................................................................... 90 DETE CTI ON TA R GET AND SA M PLE DIVERSIFICATION .................................................................................... 91 HIGH THROUGHPUT SCREENING & AUTOMATION ........................................................................................ 93 REFER ENCES ..................................................................................................................................................... 94 1 A bbre v iations AB Assay background AFNOR Ass ociation fr ancaise de normalisat ion AGFK Aspar agined, glucose, f ructose and potassium Amp Ampic illin ANOVA Analysis of variance AOAC Ass ociat ion of offic ial analytical chemists AP Alkaline phosphatase aTc A nhydrotetracycline hydrochloride ATP Adenosine triphosphate BA M Bacteriolog ical analytical manua l BLAST Basic local alignm ent se ar ch tool bp Base pairs B PW Buffer ed pep t one water BSA Bov ine ser um albumin CFU Colony forming unit CGE - PP Chip gel electrophoresis protein profiling CP4H human collagen prolyl 4 - hydroxylase DIG Digo xigen in DNA Deox yribonucleic acid DPA Dipicolin ic acid EDTA Ethylenediaminetet raacetic acid E.g . Exempli gr atia ELISA En zym e - linked immunosorbent assay ER Endoplasmic reticulum EU European U nion FAB Fragment antigen binding FISH Fluores cence in sit u hybridization FLU Fluorescence F test Fisher - Snedecor distribution GAM Goat anti - mouse GAR Goat anti - rabbit H antigen Flagellar antigen HIFa Hypo xi a - inducib le f actor HPLC High performance liquid chromatogr aphy ICAN I sothermal and chimeric primer - init i ated amplification of nucleic acids I.e . Id est Ig Immunoglobulin IPTG Is opropyl ß -D-1- thiog alactopyranoside ISO Int ernational org anization for standardization Kan Kanamycin LAMP Loop - mediat ed isothermal amp lif ication LB Luria broth mAb M onoclonal antibody MB Meat background mRNA M esseng er ribonucleic acid NASBA Nucleic acid sequence based amp lif ication NB Nutr ient broth NCBI National c enter f or biotechnology information 2 Nordval Nordic system for validation o f alternat ive microbiol ogical methods Nt Nucleotides O antigen Somatic antigen OD Optical density ORF Open reading f rame P val ue Pr obability v alue pAb Poly clonal antibody PBS Phosphate bu ff ered salin e PCR Polymerase chain reaction PDI Protein disulfide isomerase PFGE Pulse - f ield gel electrophoresis RNA Ribonucleic acid Rpm Revolutions per minute rRNA Ribosomal ribonucleic acid RSM Response surface modeling RT - q PCR Reverse transcriptas e quantitat ive PCR Rv Reverse RV Rappaport vassiliadis RVS Rappaport vassiliadis soya broth SASP Sm all ac id - soluble proteins SD Standard deviation SDS Sodium dodecyl sul f ate SHA Sandwich hybridization assay SI Supplementary information sp. Species spp. Species pluralis SSC Saline sodium citrate T BG Tetrathionate brilliant green TRAC T ranscript analysis w it h aid of affinity capture v/ v Volume per volume w/ v W eig ht p er vo l um e Deoxy nucleic aci d s A Adenine C Cytosin e G Guanine T Thymine A m ino Acids Ala A Alanine Arg R Argin ine Asn N Asparagine Asp D Aspar t ic acid Cys C Cyste ine Gln Q Glu ta min e Glu E Glut amic a cid Gly G G lycine His H Hist idine Ile I Is oleucine Leu L Leucine Lys K Lysine Me t M Methionine Phe F Phenyl alanine Pro P Proline Ser S Serine Thr T Threonine Trp W Tr yptophan T yr Y Tyrosine Val V Valine 4 A ckno w le dgme nts This w ork w as carr ied out i n th e Biopr ocess Engi neering Laborat ory at the Univer sity of Oul u and the C hair of Bi oprocess Engi neering at the Techni sche U niv ersität Ber lin. I w ant t o acknow ledge the fin ancial supp ort from the F innish Fu nding Ag ency for Technol ogy and Innov ation (TEKES), the NeoBi o and t he EN RIC H prog rams and Bi ocenter Oulu. Fir st and foremost, I w ou ld like to ex press my appr eciation and deep est grati tude to Pro f essor Peter Neu bauer f or bel ieving in me and g iving me an oppo rtunity to join hi s g reat gr oup and for introduc ing me t o the inter estin g projec t after j us t a ph one inter vi ew. I alw a ys felt v ery lucky to hav e such a fri endly , respec tf ul an d enthus ias tic super visor . I wish to tha nk Peter for his guidanc e, for bei ng so supporti v e and patien t dur ing t he w hole per iod of my w ork, and f or helpi ng me t o cont inue and f ini sh the t hesis at t he TU Berlin, ev en after a lon g break. I w ant to acknow ledge al l my co - au thors, D r. Antj e Neuba uer, Dr. Sanna T askil a, Dr. Antoni na Shvet sov a, Prof. Johana M ylly harju, Kathari na Klinz ing, M a ria Ruott inen , Dr. Mik a Tuomol a and Dr . Jari Ruuska for our ins piri ng dis cussi ons and j oint w ork. I am v ery proud to sh are my publ icatio ns w ith you. I w ould like to thank m y coll eague a nd friend from O ulu, Sa nna, for helpi ng me i n th e la b w hen I just mov ed to F inland. Y our fri endshi p and su pport me an a lot to me . It w as fun t o w ork and tr avel tog ether, share t he offic e w ith you and t alk to y ou about ever ything ! I also thank my other colleag ue and fri end, Katri n, f or being my fir st foreign fri end, tol erating my poor Engl ish and hel ping me t o adapt i n t he new envi ronment . An d my s pec ial t hanks to Chri s f or pushi ng me t o go f orw ard and hel ping w ith the thes is. I w ant to thank my dearest f riends M arina a nd Tonja for keeping m e in a g ood m ood, n ot letti ng g ive up and sho wing y our ow n good e x amples. And fin ally , I w ould lik e to t hank my fam ily , my dear husband Sergey , my w onderful so ns Vl adimir and Al exander and m y mother , f or suppor ting me thr oug hout the who le proces s. Ev en dur ing my lesser moods, yo u stood by my si de. 5 List of P ublica tions I Sandw ich E LIS A for Qua nti tative De tec ti on of Human Col l agen Pro ly l 4 - H yd r o x yl a s e Ekaterin a Osme khina , Antj e Neubauer , Kath arina Kli nzing , Joha nna M ylly harju and Peter N euba uer Microbia l Cell Fa ctories. 2010, 9 : 48 . https: //doi.or g/10.118 6/1475 - 2859 -9- 48 Licens ed u nder CC - BY 2. 0 II Quantit ative a nd sensit ive RN A bas e d detec ti on of Bacill us spor es Ekaterin a Osmekh in a , Antoni na Shv etsova, M aria Ruotti nen and Peter Neuba uer Front iers in Mi crobiolo gy . 2014 , 5:92. https: //doi.or g/10.338 9/fmicb. 2014.000 92 L icens ed u nder CC - BY 3 .0 III M odifi cati on of Buffer ed Peptone Water for I mproved Recover y of Heat - Inj ured Salm one lla Ty phi mur i um Sanna Task ila, Ekat erin a Osmekhin a , Mika T uomol a, J ari R uus ka, an d Peter Neuba uer Journal of Food Sci ence . 20 11, 76 : M1 57 – M1 62. https: //doi.or g/10.111 1/j.175 0 - 3841.2010 .020 50.x 1. General introduction GENER A L INT RO DUCTI ON INRO DUCTION Rapid and speci f ic detection and q uantification of biol ogical molecules, such as proteins and nuc leic acids, is a very wi dely used procedur e in di f ferent fields, e.g. clinica l and f ood diagnostics, environmental monitoring, basic laboratory res earch, detection of microorganisms, and d r ug production. These appro aches are i mportant for the characterization o f gene ex pression in cells on translat ional and t ranscriptional levels, an d mapping of g enes to chr omosomes. I n food technology, detection methods have allowed the rapid identification o f food - borne pathogens and contaminants. This has resulted in the w ides pread improvements in f ood quality and con trol, and have become a corners tone f or governmental authorities t o prevent, con tr ol and/or iden t ify pathogens. In the medic al f ield, DNA detection m ethods are increasingly pr oviding a solution to antibiotic res istant bacteria. The rapid overall occurrence of resistance makes these bact eria very hard to t reat with conventional antibiotics and o ften r equire detailed character ization to f in d antibiotics to which the bacteria are still suscep tible. DNA based identification o f the bacterium and its associated resistance pro files, are becomin g essential to e f fectively treat these bacterial infections. These examples show the use and widespread application o f rapid detection methods, but also illustrate the nee d for continuous development o f de tection met hods. There are mult iple factors to consider when choosing the best detection and quantitation assay, which can include the a mount of biomolecules av ai lable, their concentration in the sa mple, assay specificity, pres ence o f interfering chemicals in the solv ents used and ease, speed , cost and reliability of the assay method. PROTEIN- BA S ED DETECTI ON The first biochemical protein quanti f ication method was dev eloped in 1951 by Lowry and colleagues (Lowry et al. 1951). This method enabled the measurement of total protein c ontent in water or a very mild buffer solution by det ecting a color change o f the sample solutio n in proportion to pro tein concentr ation. The subs equent developed Bradford assay (Bradfor d 1976) is still the most popular method for quantifying pr oteins in more nativ e solutions and uses detergents or other reducing agents t o keep the prot eins soluble. These methods a re easy and f ast, but do not di stinguish between different proteins and are applicable onl y f o r total protein measure ment. Now adays the gold standard for protein quantif ication is amino acid analy sis, quantifying each individu al amino acid in a protein. This is consider ed to be the most accura te method. In proteomics, 1. General introduction 7 mass spectrometry is an o f t en used technique to measure s mall changes i n protein abundance in a complex biological system. However, these two methods are expensive, require labor intensive operation, are time cons uming and require technical ex per tise. For selective prot ein detection and measurement, the most common a nd sensitive assays are immunological - based methods that rely on the u se o f an t ibodies for protein r ecognition: quant itative enzyme - linked immunosorbent assays (ELISA), w es tern blot ting and dot blotting. The antibodies used in these methods a r e directed against con f o r mation and linear epitop es. 1.2. 1. ENZYM E - LINKE D IM MUNOSORBE NT A S S AYS (ELIS As) ELISA is a plate ba sed assay technique w hich is used for detecting and q uantifying substances such as peptides, prot eins, antibodies and hormones. ELISA methods w ere fir st described in 1971 as a n alternat ive to radioimmunoassay m ethods (Eng val l 2010; Engvall and Perlm ann 1971). Since then, ELISAs have been w idel y used for assaying soluble antigens and antibodies. The main adv antages of ELISAs include their s ensitivity, the long shelf - life, the ease o f reagent preparation, being free of radiation risks, low cost, assay speed, the reprod ucibility of the assays, and the ease by w hich the method can be converted into au tomated high throughput multiplex assays (Tighe et al. 2015) . In all ELISA methods the antigen of interest is recog nized by an antibody, the antigen - antibody - enzyme complex is bound to a solid surface (such as bottom of a wel l - plate or magnetic beads), and everything else is removed f rom the reaction through washing procedures. The amount o f bound conjugat e is visualized by incubating the solid pha se with a substrate that forms a de t ectable product when hy dr olyzed by the bound enzy me (Hor nbeck 2015) . A num ber of different enzymes have been utilized in ELISAs, including alkaline phosphatase, β - galact osidase, horseradi sh per oxidase, urease and glucoam ylase. S pecif ic subs trates, such as ortho - phenyldiam ine dihydrochloride (for peroxidase) and paranitrophenyl phosphate (for al kaline phosphatase), are used w h ich are hydrolysed by enzy m es to give colored end product. T he most used enzy me is alk aline phosphat ase because it is fast, stable, readily available and easy to use. Additionally, the substrates of al k aline phosphatase are nontoxi c and relativ e ly stable. Different ELISA types h ave been developed to increase the specificity o f m easurement: di r ect, indirect, competitive and sandw ich ELI SAs. 1.2.1.1. DI RECT ELIS A This techniq ue is the f irst ELISA method that developed, by Engvall and P er lmann in 1971 (Eng vall and Perlmann 1971). Direct ELISA is a f ast method, but has low sensitiv ity. I t is effective onl y when 1. General introduction 8 there is a high amoun t of antigens in the sample. The procedure includes coating the surface of the plate with the an t ibody or antigen ( Figure 1 .1A, st ep I), incubation w ith an enzy me tagged antibody or antigen (Figure 1 .1A, step II) and w ashing to remove the unbound antigens or antibodies from the medium. Followi ng t his, the appropriate substrate is added to the medi um to produce a si g nal through coloration (Figure 1 .1A, step I II). A colori m etric signal is m easured to determine the amou nt of the antigen or an t ibody. 1.2.1.2. I NDIRECT ELI SA The indirect ELISA was developed i n 1978 by Lindst röm and W ager (Lindstrom and W a ger 1978 ) , to measure levels of porcine IgG. In this me thod, t he wells are initially coated w ith the ant igen (Figure 1 .1B, step I), after which the diseased serum is added. This is f ollow ed up by an incubation step. An antig en – antibody complex i s formed and t he remaining liquid is washed out (Figure 1 .1B, ste p II). As a final step, the secondary enzy me - link ed antibody that r ecognizes the antibody in the serum, is added to gener ate a det ectable signal (Figure 1 .1 B, ste p III) . 1.2.1.3. COM PETITIVE ELI SA This technique, was invented in 1976 by Yorde and his cow or ker s ( Yorde et al. 1976) , s t ar ts wit h a n incubation of the sample (cont aining antigen) in a solution that contains the antibody (Figur e 1 .1 C, st e p I ) . This mix t ure is th en added to t he w ell which is coated wi th antigen ( Figure 1 .1C, step II) . T h e more the anti g en is pre sent in the sample, t he less free antibody w ill be availabl e to bind to the antig en - coa ted w ell. After the w as hing st ep, enzy m e - linked secondary antibody specif ic for the primar y antibody i s added to determine the amount o f primary antibody bound to the well (Figu re 1 .1C , step III) . The higher the concentration o f antig en in the sampl e, the l ower t he gener ated sig nal. 1.2.1.4. S A N DW ICH ELI S A This method was developed in 1977 by Kato and his cow or k ers (Kat o et al . 1977) . T he sandwich ELISA is a power f ul tool f or quant ifying proteins and quali f ying their state of activation in complex biological samples. It is the most sensitive ELISA techni que. The assay is widely used in clinical diagnost ics, f ood sample analysi s, and as a mi croarray in proteomic a pplic at ions (Nielsen and Geierstanger 2004) . The method is based on the detection of hybridization events bet ween two antibodies (capture and detect ion) and the target proteins. The capture antibody i s used to im mobilize the pro tein onto a solid 1. General introduction 9 support (Figure 1 .1D, st ep I and I I) and the de t ection antibody is recogniz ed by the enzy me - link ed secondary antibo dy (Figure 1 .1 D, ste p III and IV) . The li nked enzyme catalyz es s ubstr ate transformat ion reactions wit h generation of a de tectable signal. Fig. 1 .1 . ELISA variants. A – direct ELISA, B – i ndirect ELISA, C – competit ive ELISA, D – sand wich ELISA. 1. General introduction 10 T h e sandw ich ELISA has certain adv ant ag es w hen compar ed t o a s tandard ELISA: f irstly, it has the ability to use crude or impure samples and still selectively bind an antigen of interest; and, secondly , it has a better specificity, since the ant ibodies agai nst differ ent epitopes of a target protein are used. NUCLEIC ACID - BASED DE TECTI ON The key to the detect ion of specific nucleic ac ids is W atson - C rick base pairin g bet ween complementary strands of RNA or DNA ( W at son and Cric k 1953). A denatured sin gle stranded DNA (ssDNA) or RNA can be recognized by a comple mentary sequence of single stranded nucleic acid m olecule (probe, primer or isolated RNA or ssDN A) and attac h to form do uble - stranded molecules as dictated by complementary base pairing — a pr ocess called nuclei c acid hy br idization. This complementary nucleic acid molecule can be used for labeling, immobilizat ion or amplification of the targeted sequence to f ollow detect ion. Nucleic aci d hybrids can be f ormed between t wo strands of DNA, two strands of RNA, one strand of DNA and one st rand of RNA. A perfect match in the sequenc e o f nucleotides pro duces v ery stable nuclei c acid hybrids, w her eas one or more base mismatches impart increasing instability , resulting in w eak hybridization o f the strands. The other import ant f actors f or the hy br idization str ength ar e temperature, salts and formamide concen tration. Th e op t imum combination o f these fact ors ensures hi gh specificity and sensitivity of the nucleic acid detect ion. 1.3. 1. BLOTTING METHO DS The f irs t nucleic acid hybridization technique was devel oped by Edwin Souther n in 1975 (Southern 1975) , which is call ed Southern blotting. In this method, the target DNA digested with restriction endonucleases is separated by g el electrophoresis and a radiolabeled probe complementary to the target DNA se quence is used f or DNA r ecognition. Other blotting me thods (i.e., western blot , northern blot, eastern blot, sou thwestern blot) e mploy similar principles, but ins tead target RNA o r protein, and have later been named in reference to Edwi n Southern' s name. The detection of nuc lei c acids using the southe rn and nor thern hybridiz ati ons has the disadv ant age o f a rather low level of sensitivity, limited quant ifi cation potent ial, and a l arge time commitment, although is comparatively inexpensive. 1.3. 2. MICRO A RR A YS To measure express ion l evels o f large number o f genes, D NA mic r oarrays (Taub, DeLeo, and Thompson 1983) are r outinely used. A D NA micro a rray is a collect ion of thousands microscopic DNA 1. General introduction 11 spots (probes) speci f ic to certain g enes and at tached to a solid sur face – DNA - chip. T he DNA -chips are produced by either robotic spotting of oligonucleotides o r f ull length g ene sequences on t he chip surfac e, or by in sit u synthesizing oligonucleotides on the appropriate position on the chip. T arget RNAs or cDNAs bind to complementary probes which are us ually detec ted via fluorescent signals. Beside the transcriptional analy ses, ot her applications o f DNA microarrays include the detection of organisms by RNA/DNA sequences, comparative genomic hybridization, identif ication of gene regulator y elemen ts and monitoring o f microRNAs expr ession. The main disadvantages of m icroarray s are their high associated costs, investment in eq uip m ent and disposables, and limi tations in view of quantitative data eval uation. 1.3. 3. RNA S EQUE NCING ( RN A - seq ) Now adays , RNA sequencing based on the next - generat ion sequencing plat f or m (Mortazavi et al. 2008) is rapidly becoming the method of choice for transcriptional profiling experiments , which allo ws RNA sequencing even on sin g le cell ular level. In contrast to micr oarray technology, RNA - seq w ork s w ith a sin gle nucleotide resolution, does no t require a sequenced g enome and uses essentiall y the same p rotocol for the sequencing o f a ny species . It also has a high dy nam ic range and m inimal noise l evels (M ar inov 2017) . In addition to genome - wide analy sis of transcription, RNA - seq allo w s the identification o f alt ernative splicing an d post - tran s lational RN A editing ev ent s, and can also be used to study non - coding regions of genomes. 1.3. 4. QUA NTI T ATIVE PCR (qPCR) The most sensitive method s for individual DNA and RNA detect ion are pol ymerase chain reaction (PCR) and quantitative PCR. Both methods are able to detect single copies o f a specif ic nucleic acid. The specificity of ampli f ication is achiev ed by the use of oligonucleotide primers that hy bridize to complementary sequences on the target molecule. Depending on requirements the PCR can be performed as a quantitativ e or semi - quantit ative assay . In case o f RNA detec tion, the techni que invo lve s the conversion of mR NA to cDNA by reverse transcript ion. The cDNA is used a s a template f or the next ampli f ication steps. The ampli f ied product is detected in each reaction cycle using either fluorescent dyes incorporated in the primers , o r dyes that becom e f luorescent a f ter binding to double s tranded DNA (such as SYBR Green I). 1. General introduction 12 A major complication of RT - PCR is t he laborious and time - consuming R NA purif ication, which is necessary t o remov e any possible co nt aminants that can inhi bit the r eaction. During the p urification, parts of the molecules ar e o ften l ost, which courses di f f iculties in detecting low quantities of m R NA species. This is especially challenging for prokaryotic mRNA , whi ch is more di f f icult to pu rify compare d to eukaryotic m RNA, due to the absence o f polyadenylation. The additional purification of mRNAs increases the overall costs and the processing time. M oreover, due t o its short half - life and low stability, mRNA c an be partly degraded durin g t he purification steps and extensive centrif ugations , w hich may dramatically alter the assay results. 1.3. 5. SA NDWICH H YBRIDI Z A TION A SS A YS (SHA ) Sandwich hybridization assays (SHA) have been proved to be a g ood alternative for rapid nucleic acids quantificat ion wi thout am plification of the target (Rautio et al. 2003) . These m ethods are based on the hybridiz at ion of a ta rget RNA (or denatured DNA) with two specific oligonucleotide probes ( Fi g ur e 1 . 2 ) . A cap ture probe is used to immobilize t he tar g et on a solid su ppor t, such as microwell plate or ma gnet ic micro beads, which provide a large surface area f or nuclei c acid attachment ( W a lsh, W an g, and W ei m er 2001) . The capture probe c an be im m obilized prior, during or aft er th e hybridization. This binding betw een the probes and the surface usually takes place t hrough interact ion of bio tin attached to the oligonucleotide probe and streptavidin coated surface. The detect ion probe is l abeled wi th a marker molec ule which generates a s ignal proportional to the amount of target molecule. The f inal detection can be ba sed on time - resolv ed dyes (Hakala and Lönnberg 1997) , radioa ctive labels (Pa l va, Nyberg, and Palva 1988), or enzymatic react ion wi th generation of a f luorescent (Rautio et al. 2003) or elect rical signals (Gabig Ciminska et al. 2004 ) . Oligonucleot ide probes required for this assay can be designed for al most any RNA target and can easily be m odified f or other targets. T his m eans that the developed detection sy s tem can be applied for different organisms with just some s m all adaptations. Sandwich hybridiz at ion is relatively sensitive (10 − 16 – 10 − 15 moles of a specific target molecule) and , in contrast to R T - PCR, can be performed w ith crude biological samples without any RN A purification. The procedure does not r equire any expensiv e s pecialized equipment (the manual v er sion requires a basic t hermoshaker and a f luorescent reader). The costs include t he oligonucleotides, streptavidin - labeled magnetic beads, detect ion chemicals and plastic w ar e. 1. General introduction 13 The method has been su c cessfully applied f or the detection o f 16S rRNA from Legionella sp. in w at er samples (Les k ela et al. 2005), mycobacteria in soil (Nieminen et al. 2006) , Lactobacillus and Pediococcus in brewery y east slurr y (Huhtamella et al. 2007) , Salmonella in minced meat (Taskila et al. 2011 ) and Bacillus cereus in soil (Gabig - Ciminska et al. 2004). The method has also bee n adapted to moni tor dynamic changes of di ffer ent m RNA lev els in many microbial processes (Rautio et al. , 2003; Neubauer et al . , 2007; Soini et al. , 2008; Thieme et al . , 2008 ) . T he possibility to use variou s markers makes the method applicable for different read - out systems, such as fluorescenc e mete rs ( Rautio et al . , 2003) , ch ip - based fluor escent biosensors (W ang et al. 2013) and electrical biochip readers (Jurgen et al . 2005; El sholz et al. 2006; P ioch, Schw eder, and Jurgen 2008 ; Pioch et al. 2008; Gabig - Ciminsk a et al. 2004) . Fig. 1.2. Princ iple of bead - base d SHA In this w or k, procedures for detection and quantificat ion of proteins and R NAs in crude samples a re described, developed and optimized based on a sandwich hybridization platf orm. W e present here a sandwich ELISA for moni toring of recombinant human collagen pr olyl 4 - hydroxyl ase product ion in E. c oli cultures and a s andwich hybridization assay f or RNA - based det ection of Ba cillus s ubt ilis spores in air samples and S almonella cells in minced meat samples. 1. General introduction 14 A I M S OF THE PRESENT STUDY The aim of this w or k is to employ the s andwi ch hybridization technolog y f or R NA based detection of microorg anisms and t o moni tor recombinant protein production in E. coli cel ls. T he w or k was div ided into three projects wi th the following research goals: 1. Dev elop ment of a m et ho d f or q uant itative detection o f recombinant CP4H i n crude cell ex tracts . Special f ocus is placed o n t he devel opment of a method t h at a llows spe cif ic detect ion of the whole , and only act i ve f orm of t h e CP 4H tetramer . This is of sig nif icant import ance becau se current detection methods target single, inactive, s ubunits of CP4H . The production lev els of these indiv idual subunits vary w idel y, resulting in large inacc uracies that arise during the measurement when compared to the levels of t h e a ct ive t e t r am er f or m o f CP4H . Spe ci f ic aims t o achieve this goal include the selection of an tibodies for different subunits o f the protein, the d evelopment and optimization of t he sandw ich ELISA pr otocol for CP4H and, f inally , demons trat e t h e specificity and effectivity o f t he met hod. 2. Dev elop ment of a m e t hod f or the detection o f airborne spores. Key characteristics for the method w e aim for include f a st detection, high sensitivity (i.e. the ability to de t ect ev en very low quantities) and t he ability to detect vi able bacteria l spores f iltered from the air t o routinely monitor air q ualit y. The method development wi ll f ocus on two speci f ic parts; the activation procedure f or spore germinat ion and the e stablishment of the RNA based sandwich hy br idizat ion assay for t heir detection. Aims for the spore germination procedure include rapid recovery of spore v iability. For the SH A detec tion, aims include the achieveme nt o f high sensitivity and speci f icity , in which the a ss ay probe design will be o f significant import ance. 3. Dev elop ment of a m e t hod f or t he rapid detection of Salmonella in minced meat samples. The method development wil l comprise t he design of the Salm onella spe cif ic oligonucleotide probes f or t he RNA based sandwich hy br idization assay, optimization of the assay and applying it to the crude cell extracts of meat samples containing t h e Salmonella cultures . Special emphasis is placed on the specificity of the method, 1. General introduction 15 where f unctionality is re quired even in the presence of hi gh am o u nt s of b ac k gr ound f l or a , w hich is to be expected during rou tine detection in f ood sa mples . 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 16 S A NDWICH ELISA FOR QUA NTIT A TIVE DETECTION OF HUM A N C OLL A GEN PRO LYL 4 -HYDROXYL A SE The present work is desc r ibed here as in P ublication I (accepted v ersion) with modif ications. INTRO DUCTION W e have dev eloped the sandwich ELISA for recombinant human collagen prolyl 4 - hydroxylase (CP4H) detection in c rude cell extracts. The cap ture and detection antibodies utiliz ed in the assay were chosen to be specific to different subunits of the target protein, resulting in the effect that onl y proteins containing both subunits are sensed usin g t he assay . 2.1. 1. COLLA GEN P ROLYL 4 - HY DROXYLA SES Collagen prolyl 4 - hydro xylases (CP4Hs, EC 1. 14.11.2) play a cent ral role in the sy nthesis o f collagens and collag en - like p roteins. CP4Hs ar e located w it hin the lu men of the endoplas m ic reticulum. Their f unction is catalyzing the formation of 4 - hydroxy pr oline, which is essential f or the formation of the c ollagen triple helix , by the hy dr oxylation of prolines i n -X- Pro - Gly - s eq uences in collagens and some proteins t hat have c ollagen - like domains ( Kivirikk o and M y llyharju 1998; Kivirikko and Pihlajaniemi 1998; Myllyharju and Kivirik ko 2001) . A distinct family of cy t oplasmic prolyl 4- hydroxylases play a critical role in t he regulation o f the hy poxia - inducible tr anscription f ac t or HIFa (Bruick and McKni ght 2001; Epstein et al. 200 1) . There is no a mino acid sequence homology between the collagen and H IF CP4Hs, wi th the exception that the catalytically critical residues are conserved. The human CP4Hs are α 2 β 2 t etramers wi th a total size of 240 kDa (Figure 2. 1 ). The α subunits contain the cat alytic s ites and the β subunits keep t he protein in a soluble and ac tive st ate. The α subunit is presented as two isof orms, α(I) and α(II). The α(I) subunit consists of 517 amino acids and a signal peptide o f 17 ad ditional residues. T he α(II) subuni t consists of 514 am ino acids a nd a si g nal peptide o f 21 residues. The overall amino acid sequence similarity between the α(I) and α(II) subunits is 64%, whereas the catalytic C - terminal regions are highly identical (Helaak oski et al. 1989; Annunen et al. 1997) . B oth the α(I) and α(II) subunits associate with the same β subunit to f o rm α(I) 2 β 2 or α( II ) 2 β 2 tetramers, called t he t ype I an d t ype II enz ymes, respectively (Helaak oski et al. 1995; Annunen et al. 199 7). The ca talytic properties of the ty pe I and ty pe II CP4Hs are v ery similar, 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 17 but the enzymes differ in binding of peptide substrates and inhibitors, and in the expression patterns. Type I CP4H is t he main form in most cell types and tissues (M yll yharju, 2008) . Fig. 2. 1. Schematic repr esentation of human CP4H . The β subunit is identical t o the enzy m e and chaperone protein disulfide isomerase (PDI, EC 5 .3. 4.1) (Myllyharju, 2008), which is one of the most abun dant proteins in the endoplasmic reticulum. There, it catalyzes the disulf ide bond f ormation and rearrangement during protein folding. In the CP4H tetramer, the role o f β subuni ts is t o k eep α subunits soluble and cataly tically active, and retain them inside the endoplasmic r eticulum. The PDI contains four domains , a, b, b’ and a’. T he catalytic site s of PDI are located in a a nd a’ domains and don’ t play any role in t he CP4H te tramer activity (Vuori et al. 1992). It was show n that binding sites in the domains a, b’ and a’ are essential for the efficiency of the CP4H tetram er assembly ( Koivunen et al. 2005) . Serum CP4H levels increase in patients wi th liver cirrhosis, alcoholic hepatitis, acute hepat itis, hepatocellular carcinoma, and cholestatic di seases, and i t can b e used a s a biochemical marker for these diseases (Keiser, Vogel, and Sadikali 1972; Fuller et al. 1976; Tuderman et al. 1977; Na g ai, Kato, and Toda 19 92) . As CP4H is a potential target f or treatment o f fibrotic diseases, a bi g interest exists in recombinant expressed CP4H, whi ch can be used for detailed f unctional and structural studies. Furthermore, CP4H coex pr ession is required for recombinant coll agen production in different expression systems. An ac tive recombinant human C P4H tetramer assem bly has been su ccessfully achieved in v ar ious cell types f or above mentioned inv estig ations (Lamberg et al. 1996; Vuorela et al. 1997; John et al . 1999; Toman et al. 2000; Merle et al. 2002; Pa kkanen et al. 2003 ; Vuori et a l. 1992) . CP4H can be e f ficiently ex pr essed and assem bled in yeast , plant and animal cells, but th e 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 18 product yields are rather low . Therefore, recombinant ex pression systems using the w ell charact eriz ed and f ast growing bacterium Esch erichia c oli as a host organism w ere developed, and they are aimed at large scale produ ction of the target enz yme in high cell densit y cultivations (Neubauer, Neubauer, and My ll yharju 2005; Kersteen, Higgin, and R aines 2004; Neubauer et al. 2007) . In these production systems, CP 4H accumulation and ac t ivity was monitored by W estern blot ting, the enzyme activ it y was measured in bo th radioactive (Kiv ir ikk o and M yllyl ä 1982) and radioactivity - f r ee (Gorres and Raines 2009) assay s, and the analysis of α and β subunits expression at mRNA level with sandwi ch hybridization (Neubauer et al. 2007). But the exact lev el of produced CP4H tetramer can only be acc urately measured after HPLC purificat ion of the protein. 2.1. 2. DETECTION O F CP4H A commercially available immunoas say (Fuji Chemicals, Toyama, Japan) ( Yoshida et al. 1986) used for the C P4H analysis i n serum is based on the process o f protein capturi ng and detection via the PDI/β subunit. This meth od detects both CP4H t etramers and fr ee PDI/β s ubunits, but it is en tirely unable to dist inguish bet ween t hem. Since PDI /β subunit expression is induced be f ore the induction of α subuni t one during recom binant CP4H production in E. coli cells, t he PDI/ β subunit pr oves t o be synthesized in a large excess over the α subunit ( Neubauer, Neubauer, and M yllyharj u 2005) . T he α subunits a ggr egate immediately i f they are reco mbinantly produced alone or i f the CP4H tetr amer is dissociated (My ll yharju, 2008) . T h e P DI /β subunit is also present in excess amounts relative to t he α subunit in vivo . In the sandw ich ELI SA d esc ribed here, the capture and detect ion antibodie s are specific to the CP4H α and PDI/β s ubunits respectively. T he assay is able to detect only pr otei ns containing both subuni ts and can be performed w i th unpurified crude cell ex t ract s. T he devel oped method w as optimized by using an experimental design approach and ev aluat ed by measuring the l evels of recombinant CP4H produced. This was applied i n the optimiz ation of recom binant CP4H p r oduction in E. coli . M A TERI ALS A N D M ETHODS 2.2. 1. STRA IN AND CULTIV ATION CONDI TIONS For production of recombinant CP4H t he s train E. coli Orig ami™ ( Δ ( ar a - leu ) 7697 araD139 ΔlacX74 aphC galE galK rpsL ΔphoA PvuII phoR F '[ lac + ( lacI q ) pr o] gor522 ::Tn 10 (T c R ) trxB ::kan, Novagen) 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 19 carrying a vector for cytoplasmic expression of recombinant CP4H (pP4Hcyt) was used (Neubauer et al. 2007 ) . Sha ke flask cultivations were perf ormed in a modified LB medium containing 20 g /L tryptone, 10 g/ L yeast ex t ract , 5 g/ L NaCl and 100 mg/ L am picillin. Exponentially g rown 10 m L precultur es inoculated w it h glycerol stocks carried out in 100 mL Erlenmey er flask s at 37°C and 220 rpm on a rotary sha k er served as an inoculum f or the main cultures, which w ere perf ormed in 3 - baffled 1 L Erlenmeyer f lasks w ith a liquid volume o f 100 mL. The cultivations were carried out on a rotary shaker at 220 rpm and 25°C and were set to 20°C a f ter the second induction ( Neubauer, Neubauer, and M yllyharj u 2005). W hen the cel l density ( O D 600 ) reached 0.2, expression of the PDI/β subunit was induced with 50 μM Isopropyl ß -D-1- thiogalactopyranoside (IPTG), and w hen it reached 0.6, expression of the α subunit was induced with 200 μg/ L anhydrotetracycline hy dr ochloride (aTc, IBA Gmb H). The cultivations aimed at CP 4H production optimization w er e perform ed using gel - based EnBase ® technolog y (Biosilta Oy , Oulu, Finland). EnBase ® technology supplies the cell culture c ontinuously with glucose and thus allow s glucose lim ited fed - batch cultivation in a closed sy stem (Panula - Perälä et al. 2008; Krause et al. 2010) . Pre - cultures were prepared as described ear lier. The cultivations were carried ou t in 3 - baffled 1 L Erlenmeyer flasks w ith a liquid v olume of 1 00 mL. The gluco se- free medium containing 2.0 g/ L Na 2 SO 4 , 2.5 g/ L (NH 4 ) 2 SO 4 , 0.50 g/ L NH 4 Cl, 14.60 g K 2 HPO 4 , 3.60 g/ L NaH 2 PO 4 · 2H 2 O, 1.00 g/ L (NH 4 ) 2 -H- c it ra t e , 3 m M Mg S O 4 , 0.1 g/ L thiamine hydrochloride, 2 ml /L trace element solution (containing: 0.50 g/ L CaCl 2 · 2H 2 O, 0.18 g / L ZnSO 4 7H 2 O, 0.10 g/ L Mn S O 4 · H 2 O, 20 .1 g/ L Na 2 - ED T A, 16. 70 g /L FeCl 3 · 6H 2 O, 0.16 g/ L CuSO 4 · 5H 2 O and 0.18 g /L Co Cl 2 · 6H 2 O) and 100 mg/ L am picillin w as used. T he expression of the PDI/β s ubunit was induced wi th 50 μM IPT G w ith OD 600 r eached 3 or 10, and a fter 2 h t he expression of t he α subu nit was induced with 200 μg/ L aTc. The cultivation tempe rature of 25°C was lowered to 20 °C after the 2 nd induct ion. The cult iv ations were carried out on a rotary sha ker at 220 rpm. For some culti vations t he sha king rate was low ered to 100 rpm a f ter the 1 st induction. 2.2. 2. A N A LYSI S OF RE COMBIN A NT CP4H Cells were harvested by centrifugation at 20,000 × g for 5 min at 4°C at di fferent time points before and after the inductions. T he size of sample w as calculated so t hat i t w ould contain a q uantity cells corres ponding to 40 mL o f OD 600 = 1. The pellets were suspended in 2 mL of the ly s is buffer (0.1 M NaCl, 0.1 M g lycine, 10 μM dithiothr e itol, 10 m M T r is - HCl, pH 7 . 8) contain ing com plete E DTA - free protease inhibitor cocktail (Roche) . The suspension w as sonicat ed for 4 × 1 min at 1 min intervals on ice (Soni f ier cell disruptor B - 30 , Branson, 3 mm diameter of sonotrode, outpu t control 5) and 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 20 centrif uged a t 15,000 × g for 15 min a t 4°C. Soluble cy toplasmic fractions were collected. Recombinant CP4H was puri f ied by a pr oc edure consisting of a poly ( L - proline) affinity chromatogr aphy and anion exchange chro matography on a HiTrap Q seph ar ose colum n ( A m er s ham Biosciences) (Kivirikk o a nd Myllylä 1987) . CP4H act ivity in t he E. coli f ractions w as analysed by a me t hod based on the formation of 4 - hydroxy[ 14 C]pr oline in a [ 14 C]pr oline - labelled subst rate consisting o f non hydroxylated procollagen polypeptide chains (Kivirikk o and My llylä 1982) . Protein concentrat ions wer e determined with the RC DC Prot ein As say Kit (B io - Rad Laboratories). 2.2. 3. A NTI BO DIES An Anti - H um a n Pr o l yl -4- Hydroxylase (α) M ouse Mo noclonal A ntibody, clone 9 - 47H10 (MP Biomedicals, LLC) was used as the capture antibody (mab - α) for sand wich ELISA. Rabbit Anti - Human PDI Poly c lonal Antibody, Abcam ( pab - β A), Anti - Pr otein Disulfide Isomerase R abbit pAb , Calbiochem, Germany (pab - β B ) , P D I (H- 160) poly clonal rabbit antibody, Santa Cruz Biotechnology, Inc. (pab - β C) and Rabbit Ant i - PDI Polycl onal Antibody, Stressgen Biorea gent s (pab - β D) we r e test ed as de t ection antibodies. Alk aline Phosph atase - conjugated Aff ini Pure Goat Anti - Rabbit IgG (H+ L) (GA R - AP) and Phosphatase - conjugated AffiniPure Goa t Anti - Mouse I gG (H+L) ( GAM - AP), Jackson ImmunoResearch Laboratories, Inc. were used as t he secondary detection an tibody. The dilutions o f the antib odies were done wi th the m ain buffer (1 × PBS ( 1. 8 mM KH 2 PO 4 , 1 0 m M Na 2 HPO 4 , 140 mM NaCl, 2.7 KCl, pH 7.4) w it h 1% BSA). The capture antibody (mab - α) w as biotinyl ated with E Z - Link™ Sulfo - NHS - LC - Biotin, PI ERCE, USA. 13.3 μl o f the antibody was incubat ed w ith 1 ml of 10 mM biot in reagent, diluted in the PBS buffer , at 25°C for 1 h. The solution w as dialysed using Slide -A- Lyser ® M ini Dial ysis Unit s (10,000 M W CO), PIERCE, USA, in PBS bu ff er during 1 h. 2.2. 4. SA NDWICH E LIS A (OPTIMI ZED PRO TOCOL) The analysis w as car ried out in U - shaped trans parent 96 - well micr otit er plate s ( Greiner Bio - On e, Frick enhausen, G ermany ). T he w ells were incubat ed w it h 120 μL o f the main buffer (1 × PBS (1.8 m M K H 2 PO 4 , 10 mM Na 2 HPO 4 , 140 mM NaCl, 2.7 KCl, pH 7 .4) with 1% BSA) f or 30 min at 25°C and 700 rpm in a Thermomixer comfort (Eppendorf). After the incubation, the buffer w as discarded and the plate was allow e d to dry. The w ells were incubat ed (30 min, 25°C, 700 rpm) w ith 85 μL of 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 21 biot in - labeled capture antibody (mab - α) diluted 1:500 and 15 μL of streptavidin coated paramagnetic beads (Rautio e t al. 2003; Thieme et al. 2008), pre viously washed 3 times in the main buffer. Before adding 100 μL of t he purified protein or samples, the wel ls were w as hed once with a w ashing solution (1 × PBS containing 1 % BSA and 0 . 05 % Tween20). T he w ashing solution was incubated i n the plate for 1 min (25°C, 750 rpm) and then removed w hile the m agnetic beads w ere r etained in the wells by a mag net. After incubation of the samples (1 h, 25°C, 700 rpm), the wel ls were washed twice, 100 μL per wel l of the first detection antibody (pab - β) diluted 1:700 was added and incubated in t he plat e (1 h, 25°C, 700 rpm). 100 μL of t he sec ondary detection antibody ( GAR - AP) diluted 1:3000 was added after 3 mo re washing steps and incubated (1 h, 25°C, 700 rpm). A f ter the incubation, t he w ells were washed 3 times and t he liquid from the last washing step w as transferred into a new plate together wi th the beads. The wel ls of the new plate w er e w as hed once more. In order t o obt ain a fluorescence si g nal, 100 μL o f t he A ttoPhos ® (BBT P) subst r ate was added int o each well and the enzy matic r eaction w as allowed to t ake place w hile incubating the plate f or 20 min (37°C, 700 rpm, protected from light). T he measurement was perf ormed with 90 μL of t he liq uid f rom each well in a black microtiter plate (Op tiPl ate™ - 96F, Perk inElmer TN Lif e Sciences, USA) usin g a W allac Vic tor 2 1420 M ultilabel counter ( Perki nElmer T N Life Sciences, USA) at an ex c itation wavelength of 430/450 nm and an emission wavelength of 560 nm . 2.2. 5. EXPER IMENTA L DESIGN A ND STA TISTICA L A N A L YS IS A 3 3 f ull factorial design was used t o optimiz e the an tibodies dilutions f or C P4H sandw ich ELISA. The center points of the parameters corresponded to prev iously used concentr ations, and double diluted and double conce ntrated solutions of the a ntibodies were tested. These concen trations were logarit hmically transformed and coded as 0, - 1 and 1, respectiv ely for stati stical calculat ions ( Table 2.1). The level s of the variables and the experimental desi g n are shown in t able 2.2. I n this s t udy, the experiment design contains 60 trials including 3 center points and 1 replicate of each trial. The ratio of fluorescence signal to background obtained f or 1, 20 and 50 ng of CP4H w as select ed as a response value. The second - order polynomial model w as creat ed and analyzed using M O DDE 8 soft ware (Umetrics, Sweden). 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 22 RESULTS The main results o f this w or k are published by Osmekhina and cow ork ers . 2.3. 1. M ETHOD DEVELOPM ENT The principle o f the met hod is draw n in figure 2.2. At f irst the C P4H bi nds to capture antibodies coupled with magnetic beads. T he m onoclonal antibodies against the α subunit (mab - α) were c hosen for protein capturing t o a void the system blocking with abundant f ree PDI/β subunits. The a mount of free PDI/β subunits in CP4H producing E. c o l i cells is hig h since t he production starts with PDI/β subunits expression to keep the later expressed α subunits in a soluble form (Neubauer, Neu bauer, and M yllyharju 2005). After the bindin g step the complex is w ashed and incubat ed with de tect ion antibodies. The protein detec tion with polyclonal antibody ag ainst another CP4H subunit (PDI/β) guarantees that only proteins containing both α and PDI /β subunits wi ll be measured. G oat Anti - Rabbit IgG labeled wi th alk aline phosphatase (GA R - AP) attaches to the detection antibody, and the alkaline phosphatase catalyz es substr ate trans f or mation reactions with fluorescent products generation. T he fluoresce nce signal is proportional to the CP4H c onc entration. 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 23 Fig. 2. 2 . The pri nciple of bead - based sa ndwic h ELISA for CP 4H. CP4H tet ram er is cap tured by the m onoclo nal anti body a gainst the α subunit (m ab - α) coupled to magnetic beads. Afte r a washi ng step rabbit pol ycl onal antibody agai nst t he β subunit (pa b - β) binds to the prot ein. G oat an ti - rabbi t IgG labeled w ith alkal ine phos phatas e (GAR - AP) is used for the com plex detec tion. The A P cat al yz es reactions of su bstrate transf orm ation wit h fl uoresc ence sign al gener ation ( Osmek hina et al. 2010 ) . 2.3. 2. Mab - α A TT ACHMENT TO MAGNETIC BE A DS The mab - α labeling wi th biotin molecules and t heir attac hment onto the streptavidin bead surface were tested (Figure 2. 3 A) . Therefore streptavidin coated ma gnetic beads were incubate d wit h differ ent amoun t s of t he biotinyl ated mab - α. The antibody attached to the beads via t he biotin - streptavidin interact ion. T he formed complex was washed and dete cted wi th the Goat Anti - Mouse IgG labeled w ith alk aline phosphatase (GAM - AP). 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 24 Di fferen t ma b- α and G AM - AP antibody concentrations were used in t he ex periment. Generated f luorescent si gnals proved to be close to the maximum ever observ ed for the bead - based f luorescent assay irrespective o f the antibodies dilutions (data not shown). Bac k ground f luorescence ob t ained without adding mab - α w as 20 - 140 tim es lower compared to speci f ic signals. The best relation between the signal and background (FLU Signal / bac kgr ound ratio) w as achieved w ith the most diluted mab - α (dilution 1/500) and GAM - AP (dilution 1/2500) (Figure 2.3 B) since the bac kground f luorescence g rew wi th in creasing of the antibodies concentrations. It was deci ded to select the exact optimal antibodies conce ntrations in the sandw ich ELISA using the CP 4H te tramer as a ta rg et. 1/500 dilution of the mab - α and 1/2500 dilution f or the secondary det ection antibody were chosen for f urther experiments. Fig. 2.3 . Att achment of mab - α t o m agnetic bea ds and i nfluence of m ab - α and G AM - AP dilut ions on fluores cen t sign al. (A ) The princi ple of the test. The mab - α bin ds to magneti c beads via bi otin - st re ptav idin i nteracti on. The com plex is det ected with t he Goat A nti - Mous e IgG label ed with AP (GAM - AP). (B) The gener ated sign als obtain ed when diff erent mab - α and GAM - AP conc entrati ons were used. Th e error bars s how t he ± SD of three par allel experi m ents ( Osm ekhina et al . 20 10) . 2.3. 3. C HOOSING THE OPT IMAL p ab - β Four different polyclonal antibodies against human PD I were tested as the CP4H sandw ich ELI S A detect ion antibody ( Figure 2.4). 1 , 10 and 20 n g of purified CP4H w er e used as a target. The antibodies dilutions w er e as follows: 1/500 for mab - α and f or each pab - β, and 1/5000 f or GAR - A P 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 25 used here as a secondary detection antibody. The seconda ry antibody (GAR - AP) concentration was chosen lower than for the GAM - AP antibody used in t he previous experiment since the less amount of detectable complexes w ere expected her e. The signal / background rat ios were compared for each pab - β. This parameter was about 3 t imes higher when pab - β C was used; therefore this antibody was sele cted for f urther experiments. A big variabil ity observed for the results w as due t o clumping o f the beads i n s ome experiments . To avoid this effect, incr eas e t he efficiency of the w ashing and the sensitivity of the assay, the method was optimized i n the followed ex per iment. Fig. 2.4 . Compar ison of t he detec tio n anti bodies produce d by di fferent m anufactur ers. Puri fied CP4H was m easure d w it h the s and w ich E LISA usi ng di ffer ent pab - β antibo dies: pab - β A (Abc am ), pab - β B (Calbi ochem, Germ any), pab - β C (Santa Cruz Biotechnology , In c. ) an d p ab - β D (Str essgen Bioreag ents). The err or bars show t he ±SD of t hree par allel experi m ents ( Osm ekhina et al . 20 10) . 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 26 2.3. 4. OPTIM IZA TION OF THE METHOD The influence of di ff erent parameters (hybridization time, salt c oncentration in the w as hing buffer and shaking rat e ) was studi ed to opt imize the sensitiv it y, accuracy and reproducibility of t he assay (Figure 2. 5 ). The responses w er e present ed as absolute signal minus background signal instead of signal to background ratio used earlier in order t o visuali ze better t he e ff ect of the changed parameters. The highest fluorescent signal compared t o the bac kground lev el and the low est standard deviation w ere obtained when the protein w as incubated with each ant ibody f or 1 h. Although a lon g er hybridization (more t han 1 h) could positively ef fect on th e signal, it w as not tested, since it w ould not iceably increase the total assay tim e. The sha k ing rate duri ng t he hybridiz at ion step did not significantly influence the r esults (Figure 2. 5 A), and 700 r pm shaking ra te was selected because it prevents sedi m entation of the beads. An elev at ed concentration of NaCl in the washing buffer was expected to increase washing efficiency , but it did not have a si g nificant effect on t he signal (Figure 2. 5 B) . It w as obvious that plates need to be shaken during the w ashing phases between the hybridiz atio n steps. W ithou t shaking the beads clumped and the washing was not efficient enough causing i ncreased variation between t he replicates (Fi gure 2.5 B). Fig. 2.5 Optim iz ation of hy bridiz ation and was hing proce dures of the C P4H s andwic h ELISA. (A) Optim i z ation of the hybr idiz ation tim e. (B) Optim i z ation of the washi ng con ditions (shak ing rate and NaCl concentr ation in the washing buffer) . 1 ng of purified CP4H was u sed as target . The err or b ars s how the ± SD of thr ee par allel exper im ents ( Osm ekhina et al. 2010) . 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 27 2.3. 5. OPTIM IZA TION OF THE A NTIBODY CONCENTRA TIONS The concentr ations of the antibodies used in the sandwi ch ELISA wer e optimized using an experimental design approa ch. The full fact orial design o f response surface methodology (RSM ) was chosen to explore the optimal level of the three antibodies (Table 2.1). FLU signal / background ratio was measured with the sandwich ELISA f or 1, 2 0 and 50 n g of CP4H an d us ed as the response (Table 2.2). The da t a was f itted w ith m ultiple regression to a sec ond - order poly nom ial model e quat ion using M ODDE 8 soft ware. Table 2.1. C oded valu es for a ntib ody c oncentr ations ( Osm ekhina et al. 2010) Coded v alues Antibo dy dilut ions mab - α pab - β GAR - AP -1 1/1000 1/1000 1/ 10000 0 1/500 1/500 1/ 5000 1 1/250 1/250 1/ 2500 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 28 Table 2.2. 3 3 full f actor ial design for the antib ody concen tratio n opti miz ation Coded v alues of antib od y dilutions FLU S ignal / back gr ound 1 ng CP4 H 20 ng CP 4H 50 ng CP 4H m ab - α pab - β GAR - AP Tr . 1 Tr . 2 Tr . 1 Tr . 2 Tr . 1 Tr . 2 -1 -1 -1 4.5 1.9 25.9 11.5 61.7 16.0 -1 -1 0 3.9 2.4 31.6 9.6 44.4 9.5 -1 -1 1 3.2 3.3 20. 8 18.6 59.3 41.6 -1 0 -1 3.4 3.2 23.5 14.8 45.1 24.0 -1 0 0 3.4 2.9 28.9 19.1 43.7 35.0 -1 0 1 5.2 3.6 31.3 26.4 52.0 30.0 -1 1 -1 3.3 2.0 30.5 18.2 34.1 25.5 -1 1 0 4.1 2.8 32.0 22.9 50.9 24.8 -1 1 1 3.1 2.9 13.2 18.8 26.9 30.5 0 -1 -1 5.4 7.0 24.0 46.0 40.2 65.8 0 -1 0 9.4 7.7 58.3 50.3 100.8 75.4 0 -1 1 6.9 8.6 36.3 50.6 47.1 72.4 0 0 -1 5.4 6.7 31.4 46.1 52.7 61.9 0 0 0 7.8 6.7 36.2 49.4 47.8 74.7 0 0 1 8.4 6.5 53.8 46.2 46.9 68.4 0 0 0 6.7 6.1 48.0 44.6 75.7 79.9 0 0 0 10.0 4.7 63.7 33.8 114.1 53.4 0 0 0 9.1 7.1 55.9 44.5 64.2 66.0 0 1 -1 4.7 4.7 35.8 40.6 49.3 49.5 0 1 0 6.1 5.8 37.8 32.6 62.1 55.4 0 1 1 4.1 5.1 24.1 32.9 36.1 49.3 1 -1 -1 8.4 7.1 65.2 46.1 88.4 25.3 1 -1 0 13.5 12.2 77.6 63.7 91.4 88.2 1 -1 1 11.8 13.7 73.3 64.0 94.0 82.2 1 0 -1 10.4 11.2 65.1 63.5 76.0 88.4 1 0 0 11.4 15.5 60.5 84.6 76.6 81.2 1 0 1 15.9 10.0 90.7 69.7 96.1 78.2 1 1 -1 10.6 8.5 63.5 55.7 87.2 59.8 1 1 0 11.2 7.7 67.8 49.9 54.6 74.1 1 1 1 14.1 9.0 76.2 51.4 80.8 58.6 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 29 The analysis o f variance (ANOVA) w as applied to test the f i t of the model equation significance. The import ance o f ea ch variable and their int eractions w ere evaluated using P - val ues. P - values less than 0.05 indicate that m odel terms a r e si gnificant (5 % si g nificance level). In th e case o f measuring 1 ng of CP4H, coded val ues of each antibody dilution, interaction between mab - α and G AR - AP dilutions and pab - β dilution quadratic coded value are signi f icant model terms (Table 2.3). The model sta tist ical sig nifi c ance was chec ked by F - tes ts (Table 2.4). T he f irst F - te st, comparing modellable and unmodellable variance, was sa t isfied since its P - val ue was < 0.05. The second , lac k of f it , t es t, comparing the model and the replicat e errors wi th each ot her, was also sa tisf ied sinc e it s P - value was > 0.05. In addition, t he m odel w as evaluated using other statistical par am eters showi ng t hat the model is good (Table 2 .4). Table 2.3. ANOVA for the antib ody conce ntration m odel T erm Effec t Coefficient P - val ue Sig nificance Constant 7.46889 1.76775e - 023 Yes X 1 7.94348 3.97174 9.98059e - 021 Yes X 2 - 1.16892 - 0.584462 0.0268867 Yes X 3 1.49567 0.747834 0.00527037 Yes X 1 2 1.62401 0.812004 0.0561372 No X 2 2 - 1.7841 - 0.892051 0.0365708 Yes X 3 2 - 1.29134 - 0.645671 0.12629 No X 1 X 2 - 0.386096 - 0.193048 0.541357 No X 1 X 3 1.28773 0.643867 0.0455131 Yes X 2 X 3 - 0.735567 - 0.367783 0.246902 No A parameter is significant if its P - value is low er 0. 05. X 1 , X 2 and X 3 are coded dilutions of mab - α, pab - β and GAR - AP an t ibody, respectively. 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 30 Table 2.4. T he evalu ation of the anti body co nc entrati on m odel Parameter Value Crit ical value (to be a good model) M eaning parameters R 2 0.844 > 0. 5 Explained variation, goodness of f it p ar am et er R 2 adj 0.816 > 0. 5 Goodness of f it measure adjust ed for deg re es of freedom Q 2 0.771 > 0. 5 Predicted variation, goodness of prediction parameter R 2 - Q 2 0.073 < 0. 2 – 0.3 Appropriateness of a mo del M odel validity 0. 929 > 0. 2 5 Def ines if this is the right type of a model Reproducibility 0.797 > 0. 5 Shows replicate err or F- test for mean - squares ( P - value) 0.000 < 0. 0 5 Compares modellable and unmodellable variance Lack of fit te st ( P - value) 0.754 > 0. 0 5 Compar es model and repli cate errors with each other The models created on the basis o f signal / background ratio f or 20 and 50 ng of CP4H had similar parameters with the presented model for 1 n g of C P4H (data not show n). 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 31 Using the M O DDE 8 softw ar e optimizer tool the b est antibodies concentrations were chosen (T able 2.5). The model indi cates that the m ab - α antibody concent ration is the most important pa rameter and it has to be set at the highest lev el. Most probably , it is possible to g et a be t ter response usin g an even less dilut ed anti body, but it has no t been done because the mab - α antibody is t he m o s t expensive reagent of the assay . To reduce the method signi f icantly, the f ourt h parameter, mab - α antibody cost, was included in the optimizer. After this correction, the f ollow ing ant ibodies dilutions were selected: 1/500 f or mab - α, 1/700 for pab - β and 1/3000 for GAR - AP. Table 2.5. Optimal antibody c oncentr ati ons ( Osm ekhina et al. 20 10) Am ou nt of CP4H, ng FLU signa l / backg round Dil utions of antibody mab - α pab - β GAR - AP 1 13.37 1/250 1/ 770 1/2500 20 75.7 8 1/250 1/710 1/ 2500 50 88.7 7 1/250 1/830 1/ 3300 The r esponse surface plot shown in figure 2.6 illustrates the influence of t he parameters pab - β and GAR - AP on the signal to bac k gr ound ratio. This plot was obtained f or parameter mab - α which was f ixed a t t he dil ution 1/500. Finally t he test w as performed using the selec t ed antibodies di lutions, and the results w ere shown to be close to predicted values o f signal t o bac k ground ratio (Figur e 2. 7 ). 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 32 Fig. 2.6 . T he res ponse s urface plot s howin g the eff ects of pa b - β and GAR - AP conce ntrations . The F LU signal t o backgr ound r ati o was used as a res ponse; m ab - α conce ntration was set at the level of 0 ( diluti on 1 /500) . The anti body dilutions are pr esent ed in their co ded val ues (T able 2.1) ( O sm ekhina et al . 20 10) . 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 33 Fig. 2. 7 . Com paris on of the r esponses predic ted by the m odel and obt ained in t he experi m ent. The pur ified CP4H was used as a target. The antibo dy dilutions : mab - α 1 /5 00, pab - β 1/700, GAR - AP 1/3000. T he error bars sho w the ±SD of thr ee par allel ex perim ents ( Osm ekhina et al. 2 010) . 2.3. 6. SENSIT IVITY A ND RA NGE The assay sensitivity level and the r ange were determined using 0 – 40 ng o f pu r if i e d C P4 H t et r am er ( Fi g ur e 2. 8 ). To si mulate the natural bac kgr ound, 1 µ g of E. c ol i whole cell ex t ract was added to each sample. The detect ion limit was estimated to be 0.1 ng of CP4H. This number corresponds to signal which is above t he av er ag e o f the bac k ground si gnal for three ti mes of the blan k signal standard deviation. The dependence observ ed between the C P4H amount and t he f luorescent signal can be used for CP4H concen tr ation estimation in real samples. The r esponse w as presented as the 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 34 absolute signal minus the background si gnal in order to be able t o es timate protein amount in s amples mor e precisely. Fig. 2.8. St andard cur ve of the bea d - based sa ndwich ELI SA for CP4 H . 0 - 40 ng of puri fie d CP4H w ere use d as a tar get. T he error bars sho w the ±SD of thr ee par allel ex perim ents ( Osm ekhina et al. 2 010) . 2.3. 7. A N A LYSI S OF T HE RE COMBIN A NT CP4H EXPRESS ION IN ESCHE RICHIA COLI The effect of biological m aterial on the signal o f the sandw ich ELISA was studied by measuring the amount of CP4H in different dilutions of cell extracts of E. coli cells expressing CP4H (0.5 – 10 µg of total proteins). The cells expressing CP4H were cultivated and dis rupted as expl ained in the M ethods, diluted with the main buffer and used as a t arget f or the sandw ich ELI SA in a mount of 10 µL, corresponded to 0. 5 – 10 µg of total pr oteins. The inhibition effect was observed with increasing of the total proteins concentration in the samples ( Fig ure 2.9). To minimize this e ffect, the amoun t of total cell proteins analyzed w ith t he assay should not exceed 1 µ g. 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 35 Fig. 2.9 . Th e effect of cell lysat e on t he sign al of the s andwich ELI SA. The am ount of t he CP4H was m easure d in 0.5 - 10 µ g of extrac t of E. c oli cell s expr ess ing CP 4H. Th e error bars s how the ± SD o f thr ee parall el experi ments ( Osm ekhina et al . 2010) . The results obtained with the sandwich ELISA f or C P4H were compared with an activity assay bas ed on the format ion o f 4 - hy droxy[ 14 C]pr oline in a [ 14 C] pro line - labeled substrate consisting o f nonhydroxylated procollagen polypeptide chains (Kivirikko and My l lylä 1982) . T h e ac t i vi t y of 10 crude samples of CP4H pr oduced in E. c o l i was measured. The same samples w er e analyz ed with the sandwich ELISA f or CP 4H and the results were compared as show n in figure 2.10. A linear funct ion and co r relation coefficient of R 2 =0.986 w er e observed demonstrating high correlation between the tw o m ethods. The obse r ved correlation is vali d only for th e performed experiments, since the correlat ion bet ween specific activ ity and a mount of enz yme m ay be differ ent under different conditions such as expression lev els, cell lysis or production conditions. 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 36 Fig . 2.10 . Cor relat io n bet w een the CP4H acti vity detect ed wit h the r adio activ e as say and the CP 4H amount measur ed with t he bead - bas ed sand wich EL ISA. The plott ed amounts o f CP4H and ac tivi ty unit s were pres ented per 10 µ g of total E. col i protei n ( Osmek hina et al. 2010) . 2.3. 8. OPTIM IZA TION OF A FED - B ATCH FERMENT A TION PRO CESS FOR CP4H P RODUCTIO N IN ESCHERICHI A COLI The developed m ethod w as used in optimization o f a fed - batch fermentation process of recombinant CP4H production by E. coli . The experiment aimed at evaluating the effect of a low ered oxygen transfer rate after the f ir st induction (IP T G) and th e influence of the cell densi ty at the first induction time on the p roduc ed e nz yme amount. It w as pr oposed that a lower oxy gen transfer rate might exhib it a positive influence on the recombinant CP4H expression by E. c oli. I n co nt r as t t o sh ak e f la sk cultures where anhydrotetracyclin (a Tc) caused a long - te rm α subuni t ind uct ion, a Tc induction was only transient in bioreactor cultures w it h strong aeration, possibly by an ox idation and thereby inactivation of the induce r aTc ( Neubauer 2006). Furthermore, the production host for CP4H w as t he E. c oli Ori gami , a double mutant ( trxB gor ) , w hich also might benef it from lower culture oxygenation. This mutant is heavily disturbed in t he cy toplasmic redox system and only g rows reasonably i f combined with a supp ressor mutation in the ahpC gene. How e ver these strains show a high constitutive expression of the transcriptional regulator of the oxidativ e st ress response, OxyR (Bessette et al. 1999). Therefore, based on the comparison of earlier results for cell growth and CP4H ov erexpression in shake flas ks and in bior eactors with a v er y muc h higher oxygen transfer 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 37 rate, we have earlier suggested that a low oxy genat ion level may decrease t his oxidativ e str ess (Neubauer et al. 2007 ). In order to av oid laborious cultivations in bioreactor t he fed - batch conditions were performed in shake f lasks usin g a glucose auto - delivery sy stem ( EnBase ® ) which simulates the conditions of a glucose limit ed f e d - batch w it h c onstant glucose supply r ate ( Panula - Perälä et al. 2008) . The high value for t he sh ak ing r ate w as chosen to be 220 rpm as in the earlier experiments. The low value was set to 100 rpm a f t er the f irst induction in order to create a significant change in comparison to the high value w ithout causing a shi f t to anaerobic metabolism or a sho ck due to extreme oxy gen limit at ion. Th e shak ing rate before the inductions w as k ept at 220 rpm in t he each experiment. The induction with IPTG was performed either at an OD 600 of approximately 3 or at OD 600 = 1 0. T h e cultivation conditions for the experiments are s ummarized in table 2. 6. Each combination of parameter values w as repeated once. Table 2.6. O ptimiz atio n of CP 4H prod uction: summ ary of cul tivation conditio ns ( Osm ekhina et al. 20 10) shaki ng rate [r pm] 1 st induc tion at OD 600 = 220 (hig h level ) 3 (low level) 220 (hig h level ) 10 (hig h lev el) 220, 10 0 after 1 st induc tion (l ow level) 3 (low lev el ) 220, 10 0 after 1 st induc tion (l ow level) 10 (high l ev el) 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 38 The cell growth of the cultur es is s hown on f igure 2.11 . The higher oxygen tr ansfer led to a prolongation of the exponential phase w hich w as f ollowed by a temporary g r owth dec rease. Induct ion at OD 600 = 10 l ed t o f inal cell densi t ies that w ere on an avera ge 40% hi g her than the values achieved by earlier induction. Fig. 2. 11 . T he gro w th of E. c oli pro duce d r ecom binant CP 4H under di ffere nt cultiva tion conditi ons. T he cells were c ultiva ted wit h using E nBase ® t echnolog y, varyi ng shakin g rate and inducti on time. The arr ows indic ate the inducti ons of the PDI/β and α subuni ts expres sion w it h IPTG and aT c, res pectiv ely ( Osm ekhina et al. 201 0) . The CP4H production was monitored at 5.5, 12.5 and 24 .5 h after the first induction using the developed ELISA (Fig ure 2.12). The ex per iments with high shaking rate an d ear ly induction show ed the lowest product yield, slight ly increasing over time (black bars) . Ind ucti on w ith IPTG a t an OD 600 of 10 instead o f 3 increased the p r otein production considerably ( dark grey bars). W it h this combination of parameters (high sha king rate and lat e induction), the highest values a f ter 12.5 h in relation to the culture volume w ere achieved (lower pa r t of f ig u r e 2. 1 2 ). The cultivations w ith reduced shaking rate after carrying out the f irst induction a t OD 600 = 3 resulted in the hi ghest overall product yield relating to t he total cell p r otein amount (li ght grey ba rs, upper par t o f figure 2 .12 ). W hen the 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 39 f irs t induction w as performed at hi g h level ( O D 600 = 10) and the sha k ing rate lowered t o 100 rp m subseq uently , the second l owest product y ield in comparison to the other experiment s w as obtained (white bars). There w as a linear increase of CP4H expression per t otal protein over the cult ivation tim e. The significanc e o f the factors (shaking rate and induction time) was ev a luated st atistically usin g M ODDE 8 sof tware (Table 2.4). A positive f actor e ff ect means that the changing of the factor value leads to significant response changes. It is obv i ous that the factors were dependent of each other since there was alw a ys an interaction between t he variables. Furthermore, the continuous shaking rate of 220 rpm had a negative i nf luence on the pr otein production per cell at all sampling t im es. T he effect induction time was not ev ident in the most of cases. 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 40 Fig. 2.1 2 . CP4H pr oduction by E . c oli c ultiv ated wi th usi ng EnBas e ® techn ology, var ying shaki ng r ate an d indu ction ti me. Exp erim ents with i denti cal condi ti ons are pr esent ed as averag e values with er ror bars i ndicati ng 2 effectiv e val ues f rom the an alysi s. The upper part: the CP4H amount (ng) measur ed in 1 µ g of t otal c ell protei n. The lower part: the CP4H conce ntration ( mg p er one l it er of the c ultivati on media) ( Osmek hina et al. 20 10) . 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 41 DISCUS SI ON A method f or quantitative CP4H holoenzy m e detect ion i n crude cell ex t racts w as developed f or monitor ing o f recombinant CP4H p r oduction. CP4H coex pr ession is required for production of recombinant collagens at that the enz yme’s activ ity is a critical parameter (Lam ber g et al. 1996; Vuorela et al. 1997 ; Toman et al. 2000; M erle et al. 2002 ; Pa kkanen et al. 2003 ; Neubauer, Neubauer, and M yllyharju 2005) . Therefor e CP4 H concentration monitoring of beco mes important for the q uality control in recom binant collagen processes. The α subunit of the enzyme has a hi gh aggregation tendency, w hich may cause low amounts o f functional enz yme and conse q uently low quality of the produced collagen, i.e. temperature instability. The standard test for CP4H ac t ivity monitor ing i s measuring hy droxylation of peptides w it h radioactive substrate. W hen used with other antibodies the here dev eloped method can be pri ncipally adapted for the quantificat ion o f other protein co mplexes such as multimeric enzy m es, receptors, ion channels etc., in crude or impure samples. The limitations of the required ant ibodies avail ability can be evaded in future due to the resourc e gained wi thin the human protein atlas 2018 project (Uhlen 2010) . The developed assay is a sandw ich ELI SA based on CP 4H te tramer hy bridization with a biotin la beled capt ure antibody against its α s ubunit and a detection antibody against the PDI/β subunit. The antibodies speci f icity against the different CP 4H subunit s ensures entire holoenzy m e detection. During the as say the CP4H is captured thr ough its α subunit and all other molecules including the free PDI/β s ubunit are w as hed aw ay. As a rule, tested samples contain abundance o f PD I since it i s initially transcr ibed during the protein expression (Neubauer, Neubauer, a nd Mylly harj u 2005) and it is also pres ented in ex cess amounts in r elation to the α subunit in vivo . An experimental design approach applied for the optimal ant ibodies concentration selection is presented in this study. A f ull factorial design w as c hosen because of the possible i nteractions between the para meters. The model obt ained w as t ested by ANO VA and it proved to be statistically significant. T he model indicate d that the cap ture antibody concentration i s the mos t e ff ective parameter, which had to be set at its maxi mum level. This means that better results could be observed using the less diluted capt ure antibody , but taking into consideration its hi gh cost 1/500 mab - α dilution was used. For the other antibodies the optimal levels were select ed by the model and illustrated as a pea k on the response surface plot (Figure 2.6). The signal / background val ues predicted by the model corr elated w ell with the values w hic h were obtained i n a real assay ( Figure 2. 7 ). 2. Detection of human collagen prolyl 4- h yd r o x yl a s e 42 The sandwich ELISA sensitivity was determined to be approxi mately 0.1 ng o f CP4H an d the assay ranged between 0 and 4 0 ng of the pure protein. A hig h amount o f E. coli cell proteins had a ne g ative effect on the signals obtained with the sandwich ELISA. This could be caused by antibodies blocking with cell proteins or aggr egates o f the CP4H α and β subunits. T o minimiz e this e f fect the amount of the total cell protein used for one measure ment should not exceed 1 μg . The developed sandw ich ELISA was compared t o the current standard, a radioactive assay which was mainly used for monitoring of the recombinant CP4H assembly and activity during its production in different expression systems (Kivirikk o and M yllylä 1982). The measured concentrations o f the CP4H produced by E. c oli cells correlated well with the amount o f the enzyme activi ty (Figur e 2. 10 ). Therefore, as the sandw i ch ELISA is a quantitative and si m ple method f or C P4H measuring in crude samples, it is very suitabl e for high - throu ghput analyses instead o f the technically demanding an d time consuming radioactive assay . The sandwich ELISA was applied for CP4H level measuring in experiments aimed at optimizing of the CP4H production by E. c oli cells . T he highest speci f ic yield of active recombinant CP4H up to now i n cultivation wi th the Origami strain has been reported by Neubauer et al. (Neubauer et al . 2007 ) . Usin g the EnBase ® g lucose auto -d e live r y sys t em (Panula - Perälä et al. 2008) we w ere able to obtain f ed - batch conditions in shake flasks and succeeded to produce a high l evel of CP4H tetramer in the sha k e f lask scale (maximum product yield per culture volume w as 42.5 mg/L). It was show n that the oxygen transfer rate, the time for the f irst inducer (IP TG) addit ion and the interaction betw een these factors have a signif icant in f luence on the CP4H production. In this study t he maximum product yield w as obtained under t he following conditions: w hen the fir st induction (with IPTG) was per f orme d at an OD 600 o f 3 and the oxygen t ransfer rate was reduced after the induction. T hese results may b e treated as indi rect proof f or the supposition that the inducer aTc can be inactiv ated by a good oxygenation of the cultiv ation medium. Although not in the focus of the article, we propose that cultivations performed with aTc should be carried out under oxy g en limitation. A commercially availabl e immunoassay f or the serum CP4H detection utiliz es only antibodies specific for the PDI/β subuni t and thus detects also fr ee PDI/β - subunits (Yoshida et al. 1986 ) . A s PDI is a common and copi ous enzy me in the endoplasm ic reticulum, this method fails to analyz e t he CP4H amount or activity. In the method developed here only the CP4H tetr amers are measured due to the fact that antibodies against both subunits are used f or the protein capturing and detection. The described sandwich ELISA has a potential to be a t ool for the a ssembled CP4H quanti f ication in blood at f i broti c diseases detection. 3. RNA based detection of Bac illus spores 43 RN A B A S E D DETECT IO N OF B A CI LL US SPO RES The present work is desc r ibed here as in P ublication I I (accepted version) with modifications. INTRO DUCTION Bacterial spor es are hi ghly resist ant, dormant structures formed in response to hostile environmental conditions, which can help i n the survival of the organisms in adv er se surroundings. In its dormant stat e, the spores can surv i ve in prolonged dr ought, flooding, increased temperature and even acidic environments. Follow ing entry into suitable conditi ons, such as ambient temperatures and nutrient rich environments, sur viv ing spor es can germinate and grow out. S por e - f orming bacteria play an import ant role in food spoil age and are considered a ma jor threat in he at - tr eated food products . Spores show typical resistance to both chemicals , such as di sin f ectan t s , and physical treatments (thermal or non - thermal) used in the f ood processing industry. Some spore - forming bacteria are able to infect humans and cau se dangerous diseases, such as botulism ( Clostridium bo tulinum ), tetanus ( Clostridium t etani ), g as g a ng ren e ( Clostridium perfringens) and anthrax ( Bacillus anthr acis ). Although many of these diseases are not frequent ly e n count ered in developed countries because of the use of appropriate h ygienic practices, vaccines and ri g ht antibiotics, hi ghly virulent variants of these pathogens have emerged. This can a t least partially be explained by the overuse and misus e of antibioti cs. Of these spore - forming bacteria, Bacillus anthracis , which is the causative agent of anthrax , is t he most k n own bacter ium because of its relation to the bioterrorism attack in the United States in 2001 and t he outbreak of anthrax in f ections among injectional d rug users in Eu r ope in 2009 and 2010. 3.1. 1. BA CI LLUS A NTHR ACIS Already in the late ninete en hundreds, Robert Koc h discovered the link be tween anthrax and Bacillus anthracis and in 1881 , Louis Pasteur created the f i rst vaccine f or anthrax. Ever since, t he disease has been cha racterized in detail. How ever, especially research into the rapid detect ion o f B. anthracis has received new attent ion in r ecent decades. T his interest c an be explained by t he potential of th e u se of B. anthrac is in biological war f are and bi oterrorism. B. anthracis is the causative agent of anthrax, an o ften lethal disease in both a nimals and humans (Mock and Foue t 2001) . 3. RNA based detection of Bac illus spores 44 Anthrax has been classified into one of four syndromes based on the entry site of infection: cutaneous, gastrointestinal, inhalational, or injectional, w ith estim ated mortalities of 1%, 25 to 60%, 46%, and 33%, respectiv ely. 95% of reported anthrax cases are cutaneous (approximately 2,000 cases annually world wide), and most arise in area s where animal and w ork er v accinat ion is limited (Africa, Asia, and Eastern Europe). In case o f cutaneous anthrax, the spore s infect via tears in expo sed sk in areas. Gas tr oint estinal anthrax i s related to the ingestion of spore - contaminated meat. Correct diagnosis o f g astrointestinal anthrax is rare, but due to nons pecif ic symptomatology o f t he infection, the real incidence of this f orm o f the disease might be under estimate d. A la rg e out b r e ak of t h e gastroint estinal anthrax was report ed in T hailand, where 24 persons w h o ate contaminated beef were infected (Sweeney et al. 2011) . The largest known outbre ak of an anth rax epidemic through human inhal ation , occurred in 1979 nea r a microbiolog ical research f acil ity of the Soviet military in Sverdlovsk, Russia. This r esulted in 96 identif ied cases, o f which 64 p r oved to be f atal. The origin o f the outbreak w as determined as an airborne anthrax aerosol, coming from t he m ilita ry f ac ility (Sternbach 2003). Follow ing t his, in 2001, after the terrorist attacks on the world trade centers, a total of 22 cases of anthr ax w er e identified in the United States. B. anthracis spore powder was mailed in envelopes and almost all t he infected people w ere involved in the handl ing of po st. Out o f the 22 patients, five di ed after inhalation of the spores (Jernig an et al . 2002; Irenge and Gala 20 12) . M ore recent ly, anthrax cases emerged that spread by subcut aneous needle injection. In these case s , d ru gs (hero in ) were contaminated w ith B . a nt h r acis spor es (Berger, Kassirer, and Aran 2014) . T he f ir s t c as e of inj ectional a nthrax was des cribed in 2000 in Norway . A ma jor outbrea k of injectional anthrax followed in the Uni ted Kingdom (47 rec ognized cases and 13 deaths in S cotland, 5 ca ses and 4 deaths in E ngland) in 2009 - 2010 . So although the use o f Bacillus ant hracis and anth rax in bioterrorist attacks hav e been r are in his tory, its lethal potential emphasizes the need f or r esearch into the species an d the need for rapid and accurat e detection techniques. 3.1.1.1. B A CI LLUS A NT HR ACIS VI RULENCE B. anthracis is a l arge, Gram - positive, aerobic sp or e - forming bacillus. Several f actors contribute to the potency of Bacill us anthracis for the poten tial use as a bioterrorism a gent. First of all, t he spores are the causative a g ent o f an t hrax and are responsible f or a ll the f orms o f the disease (different symptoms of the disease are present, based on the method of spor e uptake i nt o the body). Secondly, 3. RNA based detection of Bac illus spores 45 the spores a re hi ghly infectious. Thirdly, the extreme l ongevity of the dormant B . anthracis spores keeps the bacteria potent for l ong periods of t ime. This longevity in a do r mant state is facilitat ed by the abilit y of B. anthracis spo r es to w it hstand signi f icant phy sical pr essure, chemical str esses and the absence o f nutrients f or extended periods of time. Because o f the s mall siz e and wei ght of the spores, potential outbreaks can occur and sp r ead quickly through w ater, soil and ev en air. And f inally, to cleanse or dec ont aminate exposed envi ronments is very costly and l abor intensive. W hen the spores enter a ma mmalian host, they germinate to produce vegetative bacteria that replicate and finally kill t he host. The germination can be by the hos t nutrients (such as amino acids and purine nucleosides), temperature and carbon diox ide le vel. T he vi rulenc e of the bacterium is p rovided by anthrax toxins and the capsule enc oded on two pl asmids: pXO1 and pXO 2. pX O 1 carries the genes that encode the an t hrax tox ins: protective antigen, lethal factor, and edema fact or. W hen the lethal factor and edema factor are combined w it h the prot ective antigen, they form lethal t oxi n s and edema toxin s . pXO 2 c ar r ie s t h e genes necessary f or poly -D - g lutamic acid capsule synthesis, which prevents the phagocytosis and the opsoniz ation of bact eria (Sw eene y et al. 2011) . 3.1.1.2. B A CI LLUS A NT HR ACIS REL ATIVES B. anthracis belongs to t he Bacillus cereus group, which also contains six other c losely related species: Bacillus cereus, Bacillus thuringiensis, Bacillus m ycoides, Bacil lus pseudomycoides, Bacillus weihenstephanensis and Ba cillus medusa. B. cereus is a ubi q uitous soil bacterium and opportunist ic hu m an pathogen, causing contamination problems i n the dai ry i ndustry and paper m ills. B. weihenst ephanensis is also one o f the most i mportant food pathogens. B. thuringiensis is widely use d as biological insecticide, and is spread from air planes over f ields. B. myc oides and B. pseudomycoides are ubiquitous in soil and w ater , and there a r e toxigenic strains w it hin each of these species. 3. RNA based detection of Bac illus spores 46 Fig. 3.1 . Phyloge ny of t he genus Bacillus accor ding t o (Alcaraz et al. 2010) . 3.1. 2. BA CI LLUS SUBTILI S A s a me mber o f the Bac illus genus , Bacillus subt ilis ser ves as the best studied Gram - positive, spore forming bacterium and i s t h e m ost o f ten used la bor atory model or g anism. It is considered a s the G r am - positive equival ent of Escherichia coli. B. subt ilis is cat alase - po sitive, commonly f ound in soil and obligat e aerobe; also called Bacillus g lo bigii , Hay bacillus or Grass bacill us. U nder stressful conditio ns, such as nutrient depl etion, B. subt ilis has the abil ity to f orm do rmant spores . The spo re has a speciali zed and co mplex structure, enabling the org anism to surv ive for a long t ime under harsh en vironmental conditions ( heat, radiation, desiccation, pH extremes and toxic chemicals) and in t he absence of nut rients (starvation). W h en triggered by spe cific nutrients (germinants), the spore i s capable of breaking dormancy and initiating v egetative growth. These processes are termed ger mination and outgrowth of s p or es. Because of its ability to sporulate and 3. RNA based detection of Bac illus spores 47 g er m i na t e, Ba cillus can infect and cause foo dborne diseases/food spoilage in nutr ient rich environments. Similarly, i t is via germinat ion in l ung macropha ge s that sp ores of Bac i l lus ant hracis cause pulmonary anthrax (Setlow 2003; Keijser et a l. 2007) . 3.1.2.1. SPOR ULA TION S porulation in Bacillus subt ilis h as been studied i n great detail ( F ig ure 3.2) (Tan and R amamurthi 2014) . The process is ini tiat ed by ha rsh environmental conditions, mostly starvation, which activate the Bacillus master t ranscription regulator Spo0A, through phospho rylation. T he phosphorylated Spo0A governs the t ransition from vegetative growth to s porulation. The s porulation starts with chromos ome replication and condens ation, f ollowed by anchoring of the o rigins of replication to two opposite sides of the cel l (Fig ure 3.2, Stages 0 - I). The nex t step is the asy mmet rical division o f a rod - shaped cell into t wo genetically ident ical but morp hologically distinct c ompartments: a “mother cell” and a “forespore”, h eld together by t he ex ternal cell w all (Figure 3.2, Stag e II). T hen , t he bigger mother cell “swallows” the for espore (en gulfm ent), and the engul f ing membranes fuse, producing a double membrane lay er around the f orespor e ( Figure 3.2, Sta ge III). At the end of engulfment, the forespore f loats freely i n the mother cell cy tosol. The maturation of the spore continues by the cortex and the coat assembly, whi ch protect the spore from environmental insults (Figure 3.2, Stage IV - VI). The sporulation ends when the mother cell lyses, and the mature spore is r eleased into the environment (Figure 3 .2, Stage VI I). The coat is an outer shell composed o f seventy different proteins, and the cortex is an inner shell made of specialized peptidoglycans. These two layers are separated by the outer for espore me m brane. The cortex is located betw een the t wo f orespor e membrane layers and c onsists of an inner g erm cell w a ll (similar to the ve g etative cell w all) and an outer cortex. The central part of the endospore, th e cor e, contains DNA , ribosomes and a huge amount of dipicolinic acid (DPA) chelated w ith Ca 2+ . T he coat mostly protects t he spore fr om enzymatic assaults, ensuring the r esistance to bacteriophages. The co rtex keeps the spore in a partially dehydrated state that together w ith spore mineralization and pre sence of DPA gives the resistanc e t o the hea t . At the l ast stage o f the spor ulation, the f orespore partially dehy dr ates, its D NA binds to small acid solu ble proteins (SASPs) protecting the DNA f rom damage, the chromosom e condenses into a tig ht to roidal structure, and the mother cell ly ses, releasing t h e m at ur e sp o r e (T an and Ramamurthi 2014) . 3. RNA based detection of Bac illus spores 48 Fig. 3.2 . Spor ulation of Baci llus subt ili s. 3.1.2.2. GERM INA TION Due to their high resistance to the env ironmental conditions and very low m etabolic activity , the spores ar e able to surv ive for years in their dormant state, but can rapidly acti vate and transform back into a vegetative cell under cert ain condit ions in a process called g ermination. T here are differ ent type of f actors that trigger t he spore germinat ion. In nature, the m ain factor are nutrients, such as single amino acids, sugars or purine nucleosides, or a c om bi nat ion of nutrients, e.g. asparagines, glucos e, fructose and K + (AGFK). The othe r germination agents are lysozy me, salts, high pressures, high te mperatur e, C a 2+ – DPA, e.g. released by other germinating spores, an d cationic surf actants such as dodecyl amine. The germinant m olecules ar e sensed by spor e - specific protein complexes, germination receptors, located i n the spore’s inner membrane. The mechanism of this process is not yet w ell understood. After mixi ng spores and g erminants, it takes from few minutes t o over 24 hou r s (lag period) be f ore the spore becomes co mmit ted to g erminat e, an d germinat ion w ill proceed even after removal of the germinant . The permeability o f t he inner membrane is changing, and the spore releases monov alent cations ( H + , K + and Na + ) an d Ca 2+ – DPA, and the spore c ore is p artly rehydrated (Figure 3.3, Sta ge I). Subsequently , cortex lytic enzy m es (CwlJ and Sl eB, synthesized i n sporulation) are activated and degrade the spore protective 3. RNA based detection of Bac illus spores 49 peptidog lycan cortex . This allow s t he spore core to expand and ta ke up water, increasing the spor e core volume up to 2. 5 times , leading t o a decrease of the op t ical density o f the geminated culture. The rehydration and sw elling events coincide with a loss o f dormancy and t he spore becomes heat sens itive , lik e a v eg etative cell (Figure 3.3, Sta ge II). The spore g ermination lasts about 10 minutes, and is followed by a r eactivation of t he cell metabolism and transition of t he geminated spore to a gro wing ce ll - s pore out grow t h (Fig ure 3.3 , Outgro w th). The s mall acid soluble proteins (SASPs ) that protect the DNA a r e de g raded, releasing the D NA for t ranscr iption and pr ov iding a sou rce of a mino acids f or following protein synthesis ( Setlow 2003) . Fig. 3.3. Key ev ents of spore germ ination . 3.1. 3. EXIST ING MET HODS FOR SPOR E DETECT ION Various biosensors have been developed f or the specific detection of har mful microbial spores in air samples (Gooding 2006; Fronczek and Yoon 2 015). Most methods for the detection o f Bac illu s spores have been designed f or Bacillus anthracis , a causative f actor for anthrax and a potential biological threat agent ( Edw ards, Clancy, and Baeumner 2006; Kim et a l. 2015; Irenge and G ala 3. RNA based detection of Bac illus spores 50 2012) , in order to create sensors for biol ogical warfare agents. Currently, in f ectious agents such as anthrax are usually dete cted and identified using st andard microbiological and biochemical assays, which often require isolation and/or culturing of large q uantities of the inf ectious agents, taking several days to complete. Current biosensor research aims to enh ance the accuracy, sensitivity, speed, and capabil ity of real - tim e monit oring . Being a causative a g ent, B. a nt hracis is diff icult to work with, and therefore the cl osely related species Bacillus subt ilis and B. cereus are of t e n u se d i n t he development of detection strategies ( Ara kawa et al. 2003; Stachowiak et al. 2007; Inami et al. 2009; Cheng et al. 2011) . The primary strategies for detect ion o f Bacillu s spor es include poly m erase chain reaction based techniq ues, immunoass ays, spec trometry, chromatography, and protein pro f iling (Table 3 .1). Recently also so me autonomous pa t hogen de tect ion sy stems f or aer osol collection, sample preparation and detection were developed (Hindson et al. 2005; Stachowiak et al. 2007; Re gan et al. 2008; Inami et al. 200 9) (Table 3.1). Table 3.1 . Met hods f or detect ion of Bac ill us s pores ( Osm ekhina et al. 2014) Method Sensi tivit y O rganism s Comm ents Referenc es Nucleic acid based met h ods Real - tim e PCR 1 spor e per 10 0L of air B. anthracis (Le e et al. 19 99; Mak ino and Cheun 20 03; Irenge et al. 2010; B e et al. 2013) Cultur e – base d PCR 1- 10 spores per anal ysis B. anthracis High - t hroug hp ut (Kane et al. 2009) NA SBA co upled with bios ensor 1- 10 spores per an al ysis B. anthr acis Ana lysis after 30 m inutes of germ ination (Baeum ner et al. 2004) FISH 10 3 spor es per m 3 of a ir B. anthr acis (W eerasekara et al. 2013) ICAN ( Is oth erm al and ch im eric prim er - in iti at ed 10 4 spores per anal ysis B. su bt ilis Spore germ i nation, cell l ysis and DN A (Inam i et al. 2009) 3. RNA based detection of Bac illus spores 51 am plific ation of nucle ic acids ) DN A detect ion 2h detect ion usin g m icr of luidic s ystem Autonom ous pathoge n det ectio n sy stem w ith m ultiplexe d pol y m erase ch ain reaction B. anthr acis and others (Regan et a l. 2008) RAZO R ® EX Anthr ax Air Detecti on Syst em 200 sp ores per anal ysis B. anthr acis DNA ex trac tion an d real - tim e PCR (Spauld ing et al. 2012) QCM 3.5x10 2 CFU/m l B. anthr acis QC M detec tion of single - s trand DNA obtaine d b y as y m metr ic PCR (Hao et al. 20 11) Immun oassa ys Color im etric and electroc hem ilumine scenc e imm unoas sa y 30 - 100 s pores per an al ysis B. anthr acis (Mor el et a l. 2012) ELI SA B. su bt ilis (Zhou, W irsching, and Ja nda 200 2; Ghosh, T om ar, and Go el 201 3) On - chip ELISA 10 5 spores per anal ysis B. su b til is C hem ilum inesc ence m ethod com bined with a b ioch ip. Antibo dies agains t surf ac e spore antigen were used. (Stratis - Cu llum et al. 2003) Lum inex as sa y 10 3 - 10 4 spores per m l B. anthr acis Monoc lon al antibod ies recogni ze anthrose - c on taini ng oligosac char ides on the surf ac e of B. (T am borrini et a l. 2010) 3. RNA based detection of Bac illus spores 52 anthracis endosp ores . Peptid e - Funct ion canti lever ar ra ys 10 5 spores per m l for anal y s is , B. su bt ilis 1 from 2400 spor es was c apture d (Dh ay al et al. 2006; Cam pbell and Muthar asan 2007) Chip gel electrop hores is protein pr of iling (CGE - PP) 16 par ticles per lite r 100 ce lls per anal ysis An y (adapte d for E. c oli an d B. su bt ilis ) Autonom ous m icr of luidic s ystem (Pizarr o et al. 2007; St acho wiak et al. 2 007) Multip lexe d Imm unoass a y with PCR C onfir m ation 49 spores p er lite r of air B. su bt ilis Autonom ous Detecti on of Aeroso lized Biolo gic a l A ge nts (McBr ide et al. 2003; Hi ndson et al. 2004) Others Reporter phages 1 CFU/m l B. anthr acis T he phag e s specif icall y infect B. anthracis and transduc e biolum ines cence t o the c e lls (Sharp et a l. 2016) P yr o l ys i s -- m icrom achined diff erentia l m obilit y spect rom etr y 10 3 spores per anal ysis B. anthr acis A m ic rof abricated ion m obilit y spect rom eter in com bination wit h a pattern rec ognit ion and cl assif icatio n algorithm (Krebs et al. 2006) Microc alor im etric spect oscop y 100 - 1000 spores B. subtil is, B. cereus (Arak awa et al. 2003) Laser indu ced break down spect rosc opy single p artic les B. s ubt il is In com bination with other d etect ion m ethods (H ybl, Lithgo w, and Buck le y 2003; H y bl et al. 2006) 3. RNA based detection of Bac illus spores 53 a NASBA, Nucl eic acid seq uence based am plific ati on. b FISH , Fluor escence i n situ hybridi zation. c ICAN , Isoth ermal and chi m eric prim er - initia ted am plifica tion of nucleic a cids. d ELISA , Enzy m e linke d immunos orb ent assa y . A number of direct methods are based on DNA or protein detection and cannot distinguish between viable and dead spores. Furthermore, most of them have l ow sensitiv ity or require an ampli f ication step such as PCR. 3.1. 4. RNA B ASED DETECTI ON OF B ACILLUS SPORE S RNA - based detection methods have the adv ant age to specifically analyze only v iable spores, and RNA's, especially ribosomal RNAs (rRNAs), are populated in high am ounts . T hus it is reasonable to apply RN A detection af ter the activation of RNA sy nthesis which takes place already after approximately 10 min of germinat ion (K eijse r et a l . 2007) . Mass - spectr om etr y 10 4 - 10 5 spores B. a nthrac is In com bination with other d etect ion m ethods (Lasc h et a l. 2009; Ch ena u et al. 2011; D. Li et al. 2012) Ram an s cattering 10 4 spor es Not s pecif ic Based on det ectio n of dipic oli nic ac id (Cowch er, X u, and Go odacre 2013; Ch eng et al. 2011) Optical m icr ochip arra y biose nsor 5x10 7 spor es per m l B. anthr acis and others (Bhatt a et al . 2011) 3. RNA based detection of Bac illus spores 54 M A TERI ALS A N D M ETHODS 3.2. 1. SPOR E PRE PA R A TION Bacillus subt ilis s pores w ere obt ained from cell s ( B. subt ilis 6051α, kindly provi ded by Prof. Dr. Thomas Schweder, Ernst - Mo r i t z - Arndt University of Grei f swald, Germany ) cultured in Schae ffer's sporulation m edium (8 g/ L bacto - nutrient broth , 0 .1% (w/v) KCl, 0 . 012% (w / v) M gSO 4 × 7H 2 0, 0 . 5 mM NaOH, 1 mM Ca(NO 3 ) 2 , 0.01 mM M nCl 2 , 0.001 mM FeSO 4 ) at 37°C, 20 0 r pm for 4 day s . Cultures were carried ou t in 3 - ba f f led 1 liter Erlenmeyer flasks w ith a liquid vol ume of 100 m L . A fter harvesting and extensive washing with w ater at 4°C, spores w er e inspected by phase - contrast microscopy to show that sa mples are f ree of vegetative cells (< 10%) (Figure 3. 4 ). Spores w ere ly ophilized and stored at 4°C. For c ounting, the spores were diluted in 0.9% NaCl, activ ated at 70°C f or 30 min and plated on nutrient agar pl ates (Difco, USA). Fig. 3. 4. Phase - c ontras t i m age of th e prep are d B. subti lis spores . Most of spor es are phas e - bright and c ells ar e ph ase - dark. Sporulati on fr equenc y > 90% . 3.2. 2. A C TIV A TION AND GR OWTH CONDIT IONS B. subt ilis spores were di lut ed in 0.9% NaCl and a ct ivated by t emperature treatment a t 70°C f or 30 min. Thereafter the suspensions w er e transferred into 3 - baffled 1 L Erlenmeyer flasks containing 100 m L of g er mination medium (20 g/ L tryptone, 10 g/ L yeast extract, 10 g/ L NaCl and 0.5 mM L - alanine) (Moel ler et al. 2 006). Cultures w er e carried ou t at 37°C and 200 rpm on a rotary sha k er. 3. RNA based detection of Bac illus spores 55 Culture growth was mon itored by measur ing the opt ical density at 600 nm (OD 600 , Ul t r o sp ec P r o 2100 UV/Visible Spectrophotometer, GE Healthcare, Buc kingham shire, UK). The correspondin g colony forming units (cfu/ml) were de termined by plating on nutrient agar p lates. 3.2. 3. SA MPLE PREPA R A TI ON 1 m L of the B. su btilis cultures wa s removed into pr e - cooled 1.5 ml microreaction tubes containing 0.125 × sa m ple v olume of cold 95 : 5 e t hanol:phenol (v/v) ( Thieme et a l. 2008) . T he cells w ere collected by centrifugation (15,000 × g, + 4 °C, 5 min). T he pellets were suspended in 500 μ L of 1 × TEN buffer (10 mM Tris - HCl, 1 m M EDTA, 1 00 mM NaCl, pH 8 .0) c ontain ing 1 μ L /m L RNAg uard ® RNase inhibitor (Amersham Biosciences, New Jersey, USA) and mixed by v ortexing for 1 min. After wards, c ell suspensions w er e transferred into 2 m L microcentrif uge tubes w ith a sk irt ( Greiner Bio - One GmbH, F rickenhausen, Germany ) containing 100 mg glass beads ( BioSpec Products I nc., Bartlesville, OK, USA) w i th a dia meter of 0.1 m m. Cell di sruption was perf ormed with a FastP rep ® FP120 Cell Disrupter (Bio - 101, T hermo Savant, USA ) at 4.5 m/s f or 4 × 2 5 s. The vials w ere k ept on ice for 1 min between the disruption cycles. Cell homogenates w ere centrif uged (20,000 ×g, + 4°C , 5 min) and superna tant s were snap frozen in liquid nit roge n. 3.2. 4. GENERA TION OF THE IN VITRO 16S r RNA TRANSCRIPT The target gene w as amplified by PCR using a colony of B. s ubtilis cells as a t emplate and 10 p mol of the f orward and r everse primers (W eisburg et al . 1991) (T able 3.2). The f orward primer contained at the 5′ – end the T7 promoter sequence CTA ATA C GA CTC ACT ATA GGG. The PCR conditions were as f ollow s: 3 min at 95°C; 35 cy c les of 15 s at 95°C, 3 0 s at 50°C an d 1 min 45 s at 72°C; 10 min at 72°C and a f terwards held at 4°C. PCR prod ucts were analyz ed by agarose gel electrophoresis and purified with the QIAquick PCR puri f icat ion kit (Qia gen, Hilden, Germany). Puri f i ed PCR products were quantified by absorbance measurement at 260 nm ( G eneQuant II, Viochrom Ltd., Camb ridg e, UK). 3. RNA based detection of Bac illus spores 56 Table 3. 1. Seque nces of the ol igonucl eotide pr obes used in sand w ich hybr idiz ation for the detec tion of B. s ubti lis 16S rRN A and PCR prim ers ( Osm ekhina et al . 2014) . Probe nam e Probe spe cif ic atio n Probe s equ ence 5 ’ – 3’ Loc ation of pr obe Probe m odific ation Bsub1 6Sca p c apture TG TC TCA GT C CCAGTGTGG a 319 - 3 37 5’ - b iot in Bsub1 6Sdet detect ion CGTAGGAG T CTGG G CCG a 338 - 354 3’ - digox ige nin Help 1 helper CCCCA CT GCTGCCTCC 355 - 3 70 Help 2 helper CT GGT CATCCTCT CAGA 302 - 318 Fd- T7 Forwar d PCR prim er (CT AAT ACGACT CACT ATAGGG ) AGA GTTTGATCCTGGC TCAG 10 - 29 5’ - T 7 pr om oter Rev Rever se PCR prim er CGGCT ACCTTGTT ACGACTT 1502 - 1521 NCcap Negat i ve control c aptur e probe TGTGAACT TCCAT CGGCTT GAGCC 5’ - bio t in NCdet Negat ive control detect ion prob e GAT AG T CCCT CT AAGAA GC CAT GT G 3’ - digox igenin a Nucl eotides di fferent to gener al eub acter ial p robes ar e under line d. Target RNA w as generated f rom the puri f ied PC R product by means of T7 RN A polymerase in vitr o transcript ion using the MAXI script™ T 7 - kit (Ambion, Austin, TX, USA). T he quality of the in v itro transcribed RNA was checked by the A gilent 2100 Bioanalyser and RNA 6000 Nano LabChip Ki t (Ag ilent T echnolog ies, W aldbr onn, Germany). RNA w as q uantified by a bsorbance measur emen t at 260 nm (NanoDrop ND 1 000, Thermo Fisher Scientific, USA). 3. RNA based detection of Bac illus spores 57 3.2. 5. OLIGON UCLEOT IDE PROBES The oligonucleotide probes f or B. subt ilis 16S rRNA w ere desig ned on t he basis o f general eubacterial probes w ith some modifications (Table 3.2). The ol igonucleotide probes were pu r chased from Oligomer Oy (Helsi nk i, Finland). The de t ection probe w as labeled with the D I G Oligonucleotide Ta iling Kit (Roche Diagn ostics, Mannheim, Germany ) according to the ma nufact urer's instructions. 3.2. 6. SA NDWICH H YBRIDI Z A TION ASS AY A quantitative sandw ich hybridization assay (SHA) w as used for detecting 16S rRNA molecules. The procedure was perform ed as desc ribed by Ra ut io et a l. ( Rautio et al. 2003) and Thieme et al. (T hieme e t al. 2008) w ith s mall modificat ions. SHAs w ere carr ied out in 96 - well br igh t U - shaped microplat es (Greine r- Bio - One Gm bH, Frick enhausen, Ger m any ) using a Thermomixer Comfort (Eppendorf, H amburg, Germany). The target RNA was mix ed with the 5 × SSC hybri dization buff er (0.75 M sodium chloride, 0 .075 M sodium citrate, pH 7 .0), 20% ( v/v) deioniz ed f ormamide, 3% (w /v) dextran sulphate, 0. 2% (v/v) TW EEN20 , 0.02% (v/v) Ficoll, 0.02% (w/v) polyvinyl pyrrolidone, 0.02% (w/v) bovine serum albumin, 1% (v/v) bloc king reagent (Roche, M annheim, Germany) in 100 mM m aleic acid with 150 mM NaCl (pH 7.5), 5 pmol biotin - l abeled capture probe, 1 pmol DIG - labeled detection probe, and 1 pmol of each helper probe in a t otal volume of 100 μ L . The hybridiz ation react ions w er e performed in three parallels at 750 rpm and 50°C f o r 30 min. 15 μ L o f Str eptavidi n MagneSphere ® Paramagnetic Par t icles (Promega, Madi son, WI, USA) were w ashed as recommended by the manufacturer, and added to each w ell aft er the hybridization reaction and incubated at 750 rpm and 50°C for 30 m in. Subsequently, beads were washed thr ee times with 120 μ L of a washing buffer (1% SSC, 0.1% TW EEN20) a t 25°C and 750 rp m f or 2 m in. During the washing step beads w er e kept in the wel ls with a M ag naBot ® 96 Mag netic Separation Device (Promega, Madison, W I , USA). 100 μ L of a nt i - DIG alkaline phosphatase Fab - f r ag m e nt s (Roche Diagnostics, Mannheim, Germany) were diluted with 1 × SSC, 0.1% T W E EN20 to a f inal concentration of 375 U/ L , and applied to each w ell. T he 96 - well plate w as incubated at 450 rpm and 25°C for 3 0 m in. Unbound enzy m e w as removed by w ashing three t ime s as described abov e. A f t erw ards the whole solution was transferred into new microplat es and w as hed once mo re. Finally , 100 μ L o f 1 mM AttoPhos ® f luorescent substrate (Pr omega, M adison, W I, U SA) w as added to each w ell and the enz ymatic reaction w as perform e d at 37 ° C and 750 rpm f or 20 min. 90 μ l of the content of each w ell was t ransferred into a Cost ar black 96 - we l l 3. RNA based detection of Bac illus spores 58 assay plate (Cornin g Inc., Corning, NY , USA) for the fluorescence measurement w ith a W al lac Victor multilabel counter (PerkinElmer Life Sciences , Tur ku, Finland) at an excitation w aveleng th of 430 nm and an emission w aveleng th of 560 nm . The detection limit w as defined as 3 × SD adde d to the background f luorescence. Detection limits were calculated separately for eac h SH A and signals above it w er e consid ered as pos itive. Capture and detect ion probes w hi ch are not complementary t o any RNA of B. s ubt ilis (Table 3.2, NCcap and NCdet, res pectivel y) were used as a negative control for each sample to estimate the background sig nal. 3. RNA based detection of Bac illus spores 59 RESULTS 3.3. 1. RNA B ASED D ETECTIO N OF BACILLUS SPORES The aim o f this study was the dev elopment of a diagnostic method for the detec tion of bacterial spores with Bacillus subtilis spores as emphasis. The se q uence of the steps invol ved in this procedure are shown in Figure 3. 5 . First, the spore s are activated by a heat treatment (30 min, 70°C), followed by a t ransfer into g ermination medium (K eij ser et al. 2007 ) . The d ur ation of the en r ichment cultivation w as varied depending on the initial amount o f spores. A f t er the incubation phase , collected samples were disrupted and the supernatant was utilized f or quantification of t he 16S ri bosomal RNA directly from the cell broth by using a sandwi ch hy bridization assay, without any f urther puri f ication. Fig. 3.5 . Princi ple of ai rborne spor e detecti on using a sa ndwich hybr i diz ation ass ay. Spores coll ected fr om the air ar e activ ated a nd culti vated in a ger m inati on m edium. F irs t sampl e s for an alysis c an be co llected a fter appr oximat ely 3 0 min of ger minatio n. If the am ount of spores is to o low to det ect at t his st ep, th e incubati on can be c onti nued to al lo w mul tiplicati on of th e cell s ( Os me khina et al. 2014 ) . 3. RNA based detection of Bac illus spores 60 3.3. 2. A C TIV A TION AND CULT IVATION OF BAC ILLUS SUBTILI S SPOR ES RNA - based detection of s pores is only f easibly a f ter the activation o f RNA sy nt hesis that takes place during the spore ger mination. In order to a ctivate B. sub tilis spores and ini tiate the germ ination process, dormant spores w er e exposed to hea t (70°C for 30 m in) and afterw ards cultured in the germinat ion medium at 37°C. Cell g ermination and growth w er e monitored by measuring the OD 600 (Figure 3. 6). An increase o f cell density aft er 1 h o f c ultivation indicated that germination and outgrowth were completed and p r oliferat ion be g an. Fig. 3 .6 . Grow t h cu rv e of B. subt ilis cells . Af ter he at act iva tion (70 ◦ C for 30 m in) B. subt ilis spores were inocul ated i nto a ger m ination m edium to a final conce ntrati on of 10 6 spor es/ ml ( Osm ekhina et al. 2 014) . 3.3. 3. D EVELOPMEN T OF SHA FOR B. SUBTILIS 16S rRNA For the feasibility study, oligonucleot ide probes for the detection of B. s ubtilis 16S r RNA with SH A were designed on the basis of general eubacterial probes, with some modi f ications to de tect sequenc es specific for B. s ubt ilis (Table 3.2, Figure 3.7 ). 3. RNA based detection of Bac illus spores 61 Fig. 3.7 . A fragm en t of B. subtili s 16S rRN A with oligon ucleoti de probes for SHA The probes w ere tested using an in vitr o tr anscribed fragment of B. su btilis 16S rRNA as a target molecule, which is also appli ed as a quant itative standard (Figure 3. 8 ). A concen tration of 6 × 10 7 (0.1 fmol) targ et molecules in the hybridiz at ion solution gave a si g nal that w as signif icantly abov e the detection limit. The reaction was li near over a range of nearly 3 or ders of magnitude up to approximately 3 × 10 10 i.e., 50 fm ol o f target molecules (Figure 3.9 ). Fig. 3.8. In v itr o synt hesiz ed fragm ent of B. su btilis 16 rRN A (Agil ent 2100 Bioanal ys er). Two fr agments were tr anscribed indep endent ly. The length of th e fragm ent is 1 . 5 k b, t he conce ntration of the R NA is about 30 ng/µ l (58 fm ol/µ l). 3. RNA based detection of Bac illus spores 62 Fig. 3.9 . Stan dard curv e of the B. subti lis 16S rR NA sandwich hybri di z at ion assay. The error bars s how the ±SD of t hree par allel exper iments. T he detec tion li m it is sh own as a dash ed horiz ontal line ( Osm ek hina et al. 2014) . In order to determine th e detection limit and ra ng e of the SHA for crude cell ex t racts, different dilutions of B. subtilis cell extract were analy zed. The cel ls were collected at the end o f the exponential growth phase (OD 600 = 1. 4 ), and disrupted. A total of 2. 7 × 10 2 t o 5.4 × 10 6 cells were used as target for a dilution curve (Figure 3.10). The detection limit averaged at 1 . 5 × 10 3 c ells per assay for exponentially growing B. s ubt ilis cells (7. 5 × 10 4 cells per ml of culture medium). At this growth phase, t he calculated amount of 16S rRNA was 4.8 (±0.6) × 10 4 molecules per cell. Consequently, the detection limit in crude cell extracts corresponded to 7.2 × 10 7 16S rRNA molecules. The linear phase of the assay ranged between 10 3 and 10 6 cells in the hybridi zat i o n solution. 3. RNA based detection of Bac illus spores 63 Fig. 3.10 . SHA diluti on curve for B. subti lis 16S rRN A. The diluti ons of B. subtil is crude cell extrac t, collect ed at the exponent i al gro wth phase ( OD 600 =1. 4), were used as a target. T he corr esponding number of 16S rR NA mol ecules were calc ulat ed accordi ng to the s tandar d curv e. Th e err or bars s how the ± SD of thr ee par allel experim ents a nd the detecti on l im it is sho wn as a dashed ho riz ontal line ( Os m ekhina et al. 2014 ) . In order to establish t he analy sis of cell s from e nr ichment cultures , the germination medium was inoculated with 10 5 – 10 8 act ivated B. su btilis spores per ml. A f ter 30 min o f cultivation (be f ore the f irst prolif eration), cells w er e disr upted and analy zed with SHA (Figure 3.11). I t w as possible to dete ct about 20,000 cells per assay ( 10 6 cells per ml of culture medium). This number corresponds to approximately 10 8 16S rRNA m olecules. According to t he measure ments at this sta ge of germinat ion, one B. subt ilis cell contained 5.6 (±0.9) × 10 3 16S rRNA molecules. 3. RNA based detection of Bac illus spores 64 Fig. 3 .11 . 1 6S rRN A measur ed with SHA in B. subti lis spores after their activ ati on and germ ination for 30 m in. Corr espondi ng num bers of 16S rR NA mol ecules were calculat ed accor ding to the stand ard curve (Figur e 1C). The error bar s sho w the ±SD of thre e parall el experi m ents and t he d etection l imi t is s hown as a das hed h oriz ontal line ( Osm ekhina et al . 2014) . 3.3. 4. DETECTION O F B. SUBTILIS SPORES Next, the sensitiv ity of the me thod, including activ ation, enrichment cultiv at ion and 16S rRNA analysis of B. subt ilis spores , was studied. Therefore, the germination medi um was inoculated w ith 10 1 – 10 5 activated B. subtilis spores per ml . G ermination and growth o f the cell s was m onitored by OD 600 measurement (Fig ure 3.12A). The cell s were collected, disrupt ed and analyz ed with SH A using t he probes against the16S rRNA (Figure 3.12B). Detectable signals were observed a f t er 110, 140, 245, 340, and 370 m in of cultivation f or the s amples with an initial nu mber of 10 5 , 10 4 , 10 3 , 10 2 , and 10 1 spores per ml of culture m edium, respectively. 3. RNA based detection of Bac illus spores 65 Fig. 3. 12 . Sensitivi ty of the B. subti lis spor e detec tion m ethod. ( A ) Growth curves of B. subtil is cells after s por e activ ation . The initi al num ber of spor es vari ed between 1 0 1 - 10 5 spores per ml of germinati on m edium. ( B ) Detection of B. sub tili s 16S rRNA by sand w ich hybri diz ation after enrichm ent cultiv ation. Th e error bar s show ±S D of thr ee independ ent culti vations and m easurem ents ( Osm ekhina et al . 2014 ) . 3.3. 5. ESTIMA TION OF 16S r RNA M OLECU LE LEVEL S IN B. SUBTI LIS CELLS Since the SHA sensitivity depends on the nu mber of target RNA molecules in one cell , the am ount of 16S rRNA was determined for differ ent growth phases of B. s ubt ilis cells (Fig ure 3.13). As show n earlier, one B. subtilis cell contained 5.6 (±0.9) × 10 3 16S rRNA molecu les after only 30 min of 3. RNA based detection of Bac illus spores 66 germinat ion. At the ea rly ex ponential gr owth phase, the amount of 16S rRNA molecules per cell reached a max imum (about 9 × 10 4 rRNA m olecules per cell for an OD 600 of 0.27) and 16S rR N A levels decreased in sub seq uent gr owth stages. There f ore, the analy sis is most sen sitive w ith an OD 600 v a lue of approximately 0.3. Fig. 3.13 . Levels of 16S rR NA m olecules per cell ( gray b ars) in di ff erent gr owth phas es of B. subt ilis. RNA qua ntities w er e measur ed wit h SHA. Th e growth w as foll owed by m easurin g the OD a t 600 nm ( ○) and the speci fic growth rat e was c alcul ated (blac k l ine). The dashed line s hows fi tting of the OD curve . The er ror bar s show th e ±SD of three p arallel m easurem ents ( Osm ekhina et al. 2014) . DISCUSSION T h e d et ection o f microorganisms in bioaerosols i s an important issue, both in t he rapid dete rmination of b io - warfare agents during biological attacks and in the monitoring of indoor air quality. A fast and sensitive method f or the detection of bacte rial spores w as developed in t his study. The p roc edure includes t h e act ivation of spores, their g ermination, an en richment cultivation and RNA dete c tion using a sandw ich hybridization assay. The main a dvantag es o f the dev elop ed method in compa rison to the techniques described in T able 3. 1 are sensitivity and ability to specif ically detect only viable 3. RNA based detection of Bac illus spores 67 spores. There are no sen sit ivity limitations , as t he m ethod is adaptable by the enrichment cultivation tim e. O ther procedures described, w ith s imilar sensitivit y leve l s, r equire nucleic acid ampli f ication. The RNA analy sis can be performed directly with crude cell ex tracts avoiding laborious RNA purificat ion. The w hole p rocedur e can be easily adopted for a part icular organism and performed using equipment available in a standar d biochemistry laboratory. The method w as developed f or B. subt ilis , a model o rganism capable o f spore formation , and quite abundantly present in aerosols. Since spores only contain low amounts of RNA molecules, the aim of this study w as t o initiate RNA synthesis to increase the detection sensitiv ity. At first, the spo r es are ac t ivated at 70 o C for 30 min and placed into a medium con taining L - alanine as a germ ination fact or. During germinat ion and outgrowth , the activated spores synthesize RN A, proteins, and ATP. Chromosomal replication is initiated after 30 min (Ga rrick- Silv er smit h and Tor riani 1973) and the f irst cell division takes place after appr. 7 0 m in ( Keijser et al. 2007). Depending on the initial amount of spores, cells are disrupted either directly after the germinat ion or after the enrichment cultivation requi red for the accumulatio n of necessary amoun t of RNA molecules. The presented method al low s the use of crude cells lysates as sample material for the R NA detection, and no f urther RNA purificat ion is needed ( Rautio et al. 2003; Thieme et al. 2008). W e believe that this is a strong adv antage for usin g t he sandwich hybridization assay to detect RNA levels compar ed to reverse transcri pt ase real tim e PCR. The assay is based on the hybridization event of the t arget RNA w ith two olig onucleotide probes. Alkaline phosphatase attached to one o f the probes g enerates a fluorescent signal used f or quantificat ion. The SHA has a potential to be automated, e.g. by the use o f an electr ical chip reader (Gabig - Ciminska et al. 2 004) . After the activation the spores germinated v ery quickly. The first increase in OD 600 was noticed aft e r 1 h for the cultures with an initial dose of 10 6 spores per ml. A detectable SHA signal f or thes e cultures was observed a f ter 3 0 m in of germination. Sinc e the RNA synthesis in germinating B. subt ilis spores starts within 5 min after spore activation (Matsuda and Kameyama 1986), the cells have accumulated a detectabl e amount of RNA m olecules already after 30 min. Furthermore, at this moment the spo re core i s rehydrated , ma king cells more accessi ble to disruption (Sloma and Smith 1979; Moeller et al. 2006 ) . The sensitivity of the devel oped method is not limited by enr ichment cultiv at ion , f or which the time can be adapted. Very low in itial spore amounts in the sample could be detected a fter about 5 h o f enrichment cultivation. Consequently, the maximum dur ation of the assay f or a very low amount o f spores , including spore act ivation, enrichment cultiv ation, and det ection with the SHA is abou t 8 h. 3. RNA based detection of Bac illus spores 68 The detection limit o f the SHA w as estimated as 6 × 10 7 t arget molecules, which in t he c a se of u s ing r RN A a s a t arg et , corr es ponds to about 10 3 - 10 4 c ells in a sample depending on t heir growth stage. Through the use o f i n vitro synthesized RNA probes as a s tandard for the S HA , it w as possible to estimate the amount of 16S rRNA molecules per cell. As ex pec ted, the nu m ber of molecules increased during the germination and outgrowth, reaching its maximum at the beginning of the exponential phase of grow t h, after which it slowly decreased. After 0.5 h o f germinat ion one cel l contained approximately 5.6 (± 0.9) × 10 3 16 S r RNA molecules. Ex ponentially growing cells contained about 9 × 10 4 16S rRNA molecules. These numbers correlate well w it h the known amoun t of r i b os om e s in Escher ichia coli cells, which is 6700 – 71 000 depending o n t he speci f ic grow t h rat e (Brem er 19 96) . The 16S rRNA oli g onucleotide probes used in thi s study are capable of detecting most bac teria and are relevant only for method development. Specific probes for the SHA can be designed for almost any organism or gr oups of organisms if the gene sequence is available. W e recom mend using the target probe se quences containing at least three di f ferent nucleotides co mpared to se q uences of close related or competitiv e org anisms and t hree internal mismatches f or control probes. The specificity of each desi gned probe has to be tested. The proposed procedure f or spore detectio n includes cultivation of activated spor es for approx imately 6 hours (depending of organism) with sampling every 1 - 2 hou r s for RNA analy sis. Furthermore, it is possib le to apply the p rocedure for the analy sis of mR NAs and their dynamics during germ ination. However, due to numerical advantage of riboso mes compared to a speci f ic mRNA species, the method sensi t ivity w ould be low er when mRNA is used as a t arget f or the SHA instead of rRNA . A dditionally , the analy tical error eventually woul d be higher due t o the low stability of mRNA molecules. When selecting t arget mR NAs f or specific identification of B. subt ilis , t he transcript ion prof ile of germ inated spores ( Ke ijser et al. 2007) t ogether with abundant proteins of growing Bacillus cells have to be r eviewed. Protein abundance during the exponential growth of Bacillus subt ilis has been s tudied in great detail (Buttner e t al. 2001; Eymann et al. 2004) . T he m ost abundant proteins in B. subt ilis cytosolic extracts perform mainly housekeeping functions, and include components of the translational apparatus (e.g ., translation elongation f actors, ribosom al proteins), the glycolytic pathways, the tr icarboxy l ic acid cycle, t he metabolism o f amino and nucleic acids, and protein quality control ( e.g., chaperons). The m RNAs of these proteins could be ex c ellent t ar g et s for the specific detection of B. subt ilis spo res using the presented method. 4. RNA based detection of Salmonella 69 RN A B A SE D DETE CTIO N OF SALMON E LLA The present work is desc r ibed here as in P ublication II I with modifications. This is the pee r reviewed version w it h modifications of the following article: Taskila, S, Os mekhina, E, Tuomola, M, Ruuska, J and Neubauer, P. 2011. “Modificat ion of Bu f fered Peptone W ater for Improved Recovery o f Heat - Injured Salmonella Typhi murium .” J Food Sci 76 (3): 157 – 162, w hich has been published in f inal f orm at htt ps://doi.org/10.1111/j.1750 - 3841.2010.02050.x . This art icle may be used for non - co m mercial purposes in accordance w ith W il ey Terms and Conditions for Use of Se lf - Archived Versions. INTRO DUCTION 4.1. 1. D ISEA SE Salmonella is a genus of rod - shaped Gram - n eg at ive bact eria that persists as one of the major causes for water and f ood - borne i nfect ions w orldwide. Salmonella can i nfect both animals and humans, making it a maj or public health concern. These bacteria are f acultative anaerobic and are members of the Enterobacteriaceae f amily , which includes several clinically relev ant pathogens such as Yersinia pestis , Escherich ia coli , Shige lla and Klebsiella pneumonia (Hsiao et al. 2014) . Salmonella consists of tw o species, S . bongori and S. enterica , which cu rrently include 2,500 serotypes, c apable of in f ec ting a wide range o f hosts (Popoff 2001) . S . bongori is g enerally encountered in cold - bloo ded animals and S. en t erica is re sponsible for m ost infections in w ar m blood animals and humans. In fect ions can occur all ov er the world, stemming from its abili ty to surviv e f ree in the environment in a w ide rang e of conditions f or long periods of time. Cells are capable of surviving for years i n e nvironment s w ithout food or w at er and a r e v er y resilient a g ainst pH, temperature and physical shoc ks (Xia, Zhang, and W a ng 2015; Fiskin et al . 2016) . The types of disease, and mechanisms of in f ec t ion, differ between serotypes. Even an identical isolate can manif est itself clinically diff erently, dependent on the host species, consistency of the gut f lora and competence o f immunologic responses. And infections might also vary , fr om carriage without any symptoms, t o systemic fevers that can be f atal. Salmonella is responsible for causing both Typhoidal and non - Typhoidal salmonellis (NTS). Typhoidal salmonellis causes systemic typhoid fevers, which happens a ft er Salmonella enters the bloodstream. NTS infection generally results in g astrointestinal f ood poisoning ( M. A . Gordon 2008) . 4. RNA based detection of Salmonella 70 Oral ingestion is t he main sou r ce o f Salmonella uptake from the envi ronment, most o f ten acquired by contaminated mea t or water sou r ces. However, a variety of foods can serve a s a host, and a number of illnesses is associat ed wi th consumption of such products as eggs, dairy products, tomatoes, melons, and berries ( Painter et al. 20 13) . Once ingested, the sm all percentage of cells that survive the acidic en vironment of the stomach, coloniz e t he small and l arge intestine. A c om m o n fe a t ur e of Salmonella serotypes is its intracellular g r owth, i.e. the ability to grow inside mammalian host cells. This includes epithelial cells, macrophages, lymphoid cells and other immune cells (Ry ds tröm and W i ck 2007). How ever, to ach ieve eff icient inv as ion to epithelial cells, adherence to the epithelial cells is requir ed. This is f acilitated by adhes ins s uch as f imbriae ( Bishop et al. 2008) . Although the exact mech anisms of how Salmonella invades the epithelial cel ls is not w ell unders tood, the bacteria use this to access the lymphatic system into the bloodstream ( Grant et al. 2008; M. A. Gordon 2008 ). There are around 100 genes in S almonella that are associated directl y with vi rulent f unctions. Several of these v irulence genes have been iden tif ied to be closely located on genet ic loci, w hich are termed “Pathogenicity Islands” (Holt et al . 2008). T hese pathogenicity isl ands contain the genes that encode the Type III sec retion system. This sy stem encodes a needl e- like protein appendage. Upon close proxi mity to host cells, these needl es are used t o p r obe the su rrounding and dete ct eukaryotic hosts, upon which bacterial prot eins can be directly injected into the host cells. These effector proteins can “hijack” the host cell function and enable the patho gen to surv ive, propagate and circumvent the host’ s immune response su ch as pyroptosis, a rapid caspase - 1 dependent cel l death pathway ( Bergsbaken, Fink, and Coo k son 2009) . Salmonella remains difficult to c ulture from the environment, which makes tracking the spread of an outbreak challenging. Also, many outbreaks occur in less devel oped countries, leavi ng most case s undiagnosed ( D. Gordon et al. 2011 ) . However, Salmonella is one o f four key causes o f diarr heal diseases worldwide, predicted to affect more than a 100 million cases each y ear g lobally . In the European Union, over 100,000 human cases are reported each year. European Food S afety Authority (EFSA) has es timated that the overall e conom ic burden o f huma n salmonellosis could be as high as EUR 3 bill ion a y ear . In Finland, the m ost common cause of salmonellosis are S. en terica serotypes S. T yphimurium and S. Ent erit idis. 4.1. 2. A NTI BI OTIC RE SIS TA NCE 4. RNA based detection of Salmonella 71 Antibiotics are a c ritical w eapon against invasive bacterial in f ections. Ho wever, bacteria possess many methods to rapidly acquire resistance to sub - lethal dosage of administered antibiotics. This includes shielding of antibiotic targ ets, efflux pumps that pump out internalized drugs, decreased uptake of antibiotics and ev en t he product ion of a ntibiotic degrading enzymes (Parkhill et al. 2001; Holt et al. 2008) . Fig. 4.1. G lo bal prior ity lis t of ant ibi otic resi stant patho gens. Hi ghlighted in blue are the Salm onellae (ge nus), and Enterobac teriace ae (Salm onella fam ily nam e). 4. RNA based detection of Salmonella 72 The ability of Salmonella to acquire antibiotic resistance has already been known and studied for long periods of t ime (Hensel et al. 1995). Howev er , t he wi despread administ ration of antibiotics, preventively in f eedst ock and on prescription b y pharmacies, has r esulted in the occurrence of resistant microbial at alarming rates. Antibiotic resistance is a f ast - growin g problem, where the common prescr iption of antibiotics for a wide variety of illnesses, only amplifies the rate of resistance. The W e lcome trust and the UK government have projected so me staggering numbers t o the impact o f antimicrobial resistance of 10 m illion deaths w or ldwide annually by the y ear 2050 (Tillotson 2017) . Multidrug resistance is so import ant, that the W orld Health Organization has i ssued a global prior ity patho gens list of antibiotic resistanc e ( W HO , 2017). Spearheading this list and mark ed as prio rity #1, are 3 bacter ia; Acinetobacter baumannii, Pseudomonas aeruginosa and Ent erobacterioceae ( f amily to which the genus Salmonella belongs). Criter ia for the const ruction list, that contains 12 bacter ia in total, include annual mortality rate, f requency of infection and prevalence of resistance, amon g others (Tacconelli et al. 2017) (Figure 4 .1). In contrast to Gr am - positiv e bac teria, Gram - ne gative bact eria contain the outer membrane, which explains why many conventional antibiotic treatm ents have no e f fect on them. On top o f this, many G r am - negative bacteria can rapidly conf er resistance to antibiotic treatments. The w idespr ead prescr iption o f antibiotics has led to a sha r p increase o f multidrug resistant (MD R) bacter ial strai n s isolated fr om in fected patients. There have already been several encounters of pan - drug resist ant (PDR) bacteria, that are resistant to all k nown antibiotic treatments (Bu rki 2017) . One of the reasons, as listed by t he W H O, why so m any gram - negat ive b act eria are on the global priority list (including all 3 most critical pathogens), is because o f the lack of attention f rom the medical and pharmaceutical industry. The high costs involved in the development of new antibiotics, combined with the rapid evolving of antibiotic resistance by the pathogens, have made co m mercial exploitation very di f f icult and unappeali ng. Becau se of this, t here are very few spec ializ ed methods o f therapy and treatment still relies heav ily on antibiotics. T his in turn only increases MDR, and reduces the e ffect of anti biot ics to the point w her e even “last r esort” antibio tics such as c olistin and tig ecycline loose thei r efficacy, amplifying the need for alternative treatment. The European Centre for Disease P r evention and Contr ol (ECDC ) estimates approx imately 4 milli on patients acquire healthcare associated i nfections with antimicrobial resist ance in EU member states per year, includin g 37, 000 deaths as a direct resul t (ECDC, 2018). I n the United States, the Center for Disease Control and Prevention (CDC) estimates around 2 million peopl e became in f ected wi th 4. RNA based detection of Salmonella 73 resistant bact eria in 2013, and at least 23,000 deaths as a direct result (Bar r iere 2015) . Treatment has also become increa singly expensive; patients require prolonged hos pital stay and infections increasing ly re quir e personal ized screens to identify effective antibiotics. A study in 2013 of a single hospital follow ed 1400 patient s, of w hich 188 had resistant bacteria. T heir l owest estimates o f the treatments resulted in a total o f 13,35 million dol lars (Barriere 2015). M ultiplying the costs inv ol ved for treatment by t his study by the number of patients in fect ed each year, giv es a staggering idea of the scale of this problem, and need to f ind alt ernativ e met hods of treatment. 4.1. 3. DETECTION The number o f Salmonella cells r equired to cause infections is v ery low ( Blaser and New man 1982; Fatica and Schneider 2 011) that demands the detection methods f or monitoring of Salmonella presence in food and env ironmental samples to be ex trem ely sensitive. The important characterist ics of the detection method include t he speed o f anal ysis, ease of use, the analytical per f ormance, possibility for autom ation , and reduction of total cost. In addition, other factors such as robustness, relia bilit y, t hr ough - put, overall conv enience, and the lev el of validation and standardization affect the applicability of the method. Tr aditional me thods for testing o f food and environment al samples f or the pr esence o f Salmonella include the following steps: (i) nonselective pre - enr ichment o f a defined weigh or volume o f the sample (e.g., 25 g of minced meat) (ii) selective enrichment (iii) plating on selective a g ar (i v) biochemical and serological confirmation The pre - enrichment and enrichment steps are ne cessary to incr ease the num ber of target cells for a following detection procedure. None of the cur rently av ailable met hods can prove the demande d by EU absence of Salmonella c ells in 10 - 25 g of food w it hout th e enrichment c ultivation. The m ajor challenges in the enrichment are the low c oncentration of the target cell s in the samples and the poor physiological conditions of the cells due to environmental stress f actors, such as hi gh tempe ratu re, os motic s tre ss, o r low pH . Sublethal heat inj ury has been reported to change the metabolism of bac t eria by causing the de gr adation o f riboso mal DNA and RNA (T omlins and Ordal 1971; Tomlins, Vaaler, and Ordal 1972; Gomez et al. 1973; Gomez and S inskey 1975; Chambliss et al. 2006), which can lead to failure in recovery o f the cells and therefore, false negative results in 4. RNA based detection of Salmonella 74 detect ion. Based on the literature, the recovery o f heat - injured cells depe nds on the mediu m , and especially the use of complex media has been reported to extend t he lag times in the cultures (Gomez and Sinskey 1973; M at tick et al. 2001; Zhao and D oyle 2001). During the primary recovery in nonselective medium, the Salmonella cells should achieve suff icient concentration to tolerat e the toxicity of the selectiv e c onditions in the second ar y culture (Chen, Fras er, and Yamazaki 1993) . How ever, as the incubation in the universal enrichment broths supports also the gr owth of the background f lora (Beckers et al. 1987), it is bene ficial already to intr oduce s ome selectivity i nto the primar y nonselective me dium by for example by diffusion of (Baylis, M acPhee, and Betts 2000; Kang 2002) or increasing the incubation temperature ( Krascsenicsova, Kaclikova, and Kuchta 2006) . In the EU, the reference detection me thods are published by the International Organiz ation f or Standardization (ISO). The re ference procedure f or the detection o f food b orne Salmonella includes non - selective enrichment cultivation in buffered peptone w at er (BPW) broth for 16 – 20 hours , selective enrichment cultivation in tw o selective media and incubation on two di ff erent selective agar plates for isolation of colo nies ( I SO , 2017). The colonies are then identi f ied by m eans of biochemical test s. This procedu re takes at least 72 hours to complete. In the U nited States (US), the g overnment publishes the Bac teriological Analytical M anual (BAM) whi ch includes a c ollection of recommended procedures for the detection of pathogens and toxins in f oods. The BAM pr ocedure f or the detection of f ood - borne Salmonella includes non - selective enrichment cultivation in nutr ient broth (NB) for 16 hours and selectiv e enrichment cultivation in either Rappaport – Vassiliadis (RV) or te trathionate brilliant green (TBG) b roth for an additional 16 ho ur s (Andrews and Hammack 2007) . T he colonies are isolated by using selective agar plates and identif ied biochemically. In the past three decades, there have been increasing efforts towards developi ng r apid methods f or identif ying and de t ecting Salmonella speci f ically in food products (Bell et al. 2016; W u 2017) . Mos t of them still i nvolve the enrichment cultivation steps because o f the strict sensitivity requirements. Some of them are l isted below. 4.1.3.1. BI OCHEMI CAL TESTS The biochemical tests i nclude fermentation of glucose, lysine decarboxy lase ac tivity, negative urease reaction, detection o f H 2 S production, and f ermentation of dulci tol. The con f o r mation is performed using serological tests typically w ith antisera for detection of f lag ellar ( H) and soma tic (O) antig ens. P ositive isolates o f ten require f urt her serotyping and are se nt for iden t ification using techniq ues such as phage t yping, antibiotic susceptibility and pul sed - field g el electrophoresis (P F GE) . 4. RNA based detection of Salmonella 75 4.1.3.2. I MMUNO A SS A Y D ETE CTION Non - competitive ELISA is the most commonly us ed assay immunol og y - based assay f o r the detect ion o f Salmonella spp. In the ELISA as say , a specif ic Sal monella antigen is bound to the appropriat e antibody linked t o a solid matrix. The formation of the antigen - antibody complex can be measured through the change in color or i ncrease o f f luorescence caused by t he enzy matic cleavage of a chromogenic or fluorescent substrate that co rresponds to the concentration o f the antigen and the pres ence o f Salmonella. The different ELISA systems have been developed and commercially available in k it form: Assurance GDS™ for Salmonella (BioC ontrol Systems, Inc., Bell evue, W A) , TECRA Salmonella (Tecra International Pty Ltd, French Forest, New South W ales, Australia), Salmonella ELISA Test SE LECTA/O PTIM A ( Bioline APS, Den m ark), and Vitek Immuno D iagnostic Assay Sy stem ( VIDAS) ( BioMerieus, Hazelw ood, MO). 4.1.3.3. PCR/ qPCR - B ASED DETECTION The use of PCR based applications has been quite established in the d etect ion of Salmonella . In this, a variety of genes that are v er y specific to Salmonella cells are used as a target. Target genes have included genes t hat encode t oxins, enzy mes, rRNA g enes and re petitive elements, al l solely encountered in Salm onella (Levin 2009). r RNA genes are particularly int eresting targets, which stems from the wi de availabi lity of r RNA sequencing data. A p r oblem w it h PCR based app roaches is that there is a larg e pe r centage of mic r obial flora t hat have nev er been anal yzed, for instance due to di f f iculties in culturing conditions. The lac k of informat ion on se quences means that primers for target genes might very w e ll also be present in the unidentified species, r esulting in f alse positives (Marchesi and Ravel 2015) . To overcome this however, several specific genes can be targeted simultaneousl y, significantly reducing the chance of false positive reaction s. The detection is o f ten combined w ith an enrichment cultiv ation and h as been used to detect Salmonella in meat samples (Regan et al . 2008; M cG uinness et al . 2009) and eggs (Malorny, Bunge, and Helmuth 2007; S. Li et al. 2010). This method can be adapted for rout ine screenin g and provide results within 24 hou r s, wi th detection limits ranging f rom 10 3 – 10 4 cells per mL. Howev er , a significant drawback is that t he method is often not properly validated against the reference method, resulting in significant in performance variations between assays. The performance of the me thod has currently only been v alidated on a few ex amples, whereas Salmonel la infections can reside from a wide range o f environments/sources. Also, the e f ficiency of PCR am plification can dec r ease significant ly w hen in lo w purities of tar get DNA, illustrating the need for D NA clean - ups for effect ive detect ion (Jyoti e t al. 2011) . 4. RNA based detection of Salmonella 76 4.1.3.4. NUCLEUI C A CID - B ASED DETECTION There are also several other DNA - based m ethods dev eloped t o de t ect Salmonella . On e of the se more widespread methods is Fluorescence in Situ Hybridization (FI SH), w h ich ut ilizes fluorescent probes that can spe cifically target regions w ith a high degree o f sequence complementarity (Feder et al. 2001). T he combi nation of PCR t ogether with hybridization, can r esulted in an increased sensitivity of detection and has been reported to e nable detection times of less than 24 h our s in f ood samples ( Fang et al. 20 03) . T h er e ar e also various other DNA - based detection m ethods, such as isotherm al nuclei c acid s equence - based ampli f icat ion (NASBA ), LAMP and different hy br idization assays used, although be it to a lesser ex tent (Almeida et al. 2010) . 4.1.3.5. SEQ UENCING Significant efforts and advances in sequencing capabilities have m ade techniques li k e nex t generation sequencing (NGS) and w hole g enome sequencing ( W GS) more feasible to utiliz e in pathogen identification (Rebecca e t al. 2015) . These methods enable the whole pathogen genome to be sequenced in a matter of hours when coupled to pow er f ul and automated bioin f o rm at i cs analysis tools. The high lev el of detailed in f ormat ion can be used f o r identification and charact eriz ation. However, this method would als o enable identification the source of the pathogen, and could provi de great i nsig ht into how the disease sp reads. Currently, thi s is a pr ocess w hich is largely unknown due to difficulties in e f f icient cul ture methods of env ironmental isolates. This method could also be u sed to track and study g enetic changes occurring ov er t ime i n Salmonella , sp ecif ically relate d t o the ac quisition of antibiotic resistance ( Zhang et al. 2015 ) . Despite its potential, NGS faces currently faces sev eral drawbacks. Sequencing efforts require relatively high amounts of DNA, of w hich t he purity is important. Also, the presence of f oreig n DNA sources wil l significant ly c omplicate analysis. Fully opt imized and auto mated bioin f ormatics analysis is in many projects not established yet, and analy sis of t he large amounts of sequencing data remains labor intensive and time consu ming (Tyson et al. 2015 ) . In this study, the commonly used nonselective m edium buffered peptone w at er (BPW ) and the enrichment cultivation temperature were optimized by means of response s urface m odeling ( RSM ) in order to improve the r ecovery of heat - injured Salmonella Typhimurium. BP W was supplem ented with the enzyme - controlled substrate delivery system EnBase® (Panula - Perälä et al. 2008) and a 4. RNA based detection of Salmonella 77 Salmonella selective iron supply, f errioxamine E (Kingsley et al. 1999) . The modificat ions were shown to increase the c e ll densities in single strain cultures of Salmonel la. Furt hermore, the quantificat ion of 23S rRN A by a sandwich hy br idizat ion assay (SHA) (Rautio et al. 2003; Leskela et al. 2005; Huhtamella et al . 2007) proved the bene ficial effect of the modifications on the recovery o f heat - injured S. Typhimurium in presence of m inced meat bac kgr ound. The results presented in this study can be used for dev elopment of analytical procedures with higher accuracy o f detect ion. Fig. 4.2 . T he prop osed sch eme for the detec tion of S alm onella in foods by means of 2 3S rRN A targeting s and w ich hybr idiz ation assay (Taskil a et al. 2011) . M A TERI ALS A N D M ETHODS 4.2. 1. ST RA INS Salmonella Typhimurium DSM554 w as k indly pro vided by Scanbec GmbH (Halle, Germany). 4.2. 2. CULTIVATION MEDI A AND CONDITI ONS 4. RNA based detection of Salmonella 78 The precultures were pr epared from glycerol stocks in 5 mL o f BP W (Oxoid Ltd., Ca mbridge, U.K.) in 125 - mL ba f f led E r lenmeyer f l asks and i ncubated at 37° C and 200 rpm (In f ors M initron, Infors AG, Bottmingen, Sw itzerland) . The enrichment cultures for sampling w ere pr epared in 2 m L o f BP W o r Rappaport - Vassiliadis soy broth (RVS, Oxoid Ltd.) in 24 - w ell deep well plates (BioSilta Oy, Oulu, Finland) covered with plastic cover. The enzyme - based substrate delivery system EnBase ® Flo (BioSilta Oy) was used in this study. For op timization of the media, di ff erent concentrations o f the EnBase® soluble carbon source, Enz I’m (BioSilta Oy), and f errioxamine E (Sigma - Aldr ich, Helsink i, Finland) were tested. The cultur es were incubated in an Infors Minitron incubator (Infor s AG) a t differ ent temperatures w it hout shaking. The growth in the cultures was monitored by measuring the absorbance in 100 - μ L s amples at 490 nm (Victor3 Multiw e ll Plate Reader, Perkin Elmer, T urku, Finland) in 96 - w ell microplates. The corr esponding cell concentration (CFU/mL) w as detected by plating on nutrient agar - p lates (0.5% peptone, 0.3% beef ex tract, 1.5% agar). 4.2. 3. P R E P AR AT IO N O F H E AT - INJURE D CEL L S Heat - injured cells were prepared from exponentially gr owi ng pr ecultures in BP W . Al iquots of 1 mL were incubated in closed 1.5 - mL micr o react ion tubes (Epp endorf, Hambur g, Germany) a t 55° C under constant shaking conditions at 500 rpm ( Thermomixer Comfort, Eppendorf) for 15 m in. A fter incubation, the cells were se rially diluted in 0 . 9% NaCl, and heat injury w as confirmed by plat ing 3 replicate s on xy lose lysine deoxycholate - agar plates (Fluka XLD A g ar, Sigma - Aldric h). 4.2. 4. M EA T S A MPLES The meat sa mples w er e prepar ed f rom 4 di fferent types o f Finnish comme rcial minced meat. M eat portions of 25 g and 225 mL o f medium were hom ogenized in Stomacher® bags using a S tom acher® 400 laboratory blender (Sew ar d, W orthing, U.K.) at normal speed f or 1 mi n. For each cultur e, 2 - mL portions of suspension were tr ansferred from the liquid compartment o f the bag to the deep - we l l plate and approximately 60 Sal monella cells were added. 4.2. 5. GROWTH CURVE S The growth curves o f the cultures , based on optical densities at 490 n m (OD 490 ), were f itted into the bacterial growth model suggested by Barany i and Roberts (1994) using DMFit softw are (I nst. of Food Research, U.K.) (Baranyi and Tamplin 2004). T he corresponding l ag times w er e calculated by the soft ware. 4. RNA based detection of Salmonella 79 4.2. 6. CELL LYSIS Cell cult ur e samples o f 1 mL w ere c ollected in 2 m L Eppendorf tubes containing 0.125 × sample volume of cold ethanol/phenol (95/5 v/v). T he cells were collected using centrifugation at 15000 × g at +4° C for 5 min. T he pellet w as s uspended in 500 μ L v olume of 1 × T EN (10 mM Tris - HCl, 1 mM EDTA, and 100 mM NaCl, pH 8.0) bu f fer containing 1 μ L/mL o f RNAguard RNase inhibitor (Amersham Pharmacia Biotech Inc., N.J., U.S.A .) and 100 mg of 0.1 mm glass beads (BioSpec Products Inc., Bartlesv ille, Okla., U.S.A.). Sampl es were dis rupted using t he FastPrep FP120 Cell Disrupter ( Thermo Electron, W a ltham, M as s., U.S.A. ) w ith 4.5 m/s speed for 4 × 25 s. The samples were cooled on ice between the sequences. The cell homogenates were centrifuged at 15000 × g at 4 ◦ C for 5 min, and t he supernatants w er e subsequently frozen using liquid nitrogen in al iquots. 4.2. 7. OLIGON UCLEOT IDE PROB E S FOR SHA The oligonucleotide pr obes f or detection of S. enterica 23S rRNA ( Table 4. 1) were desi gned using the Clone M anag er software (Scientif ic & Educational Software, Ca ry, N.C ., U.S.A.) and pur c h as e d from Oligomer Oy (Helsink i, Finland). The Sal3_1816 probe is the extende d Sal3 probe u s ed in the FISH method f or detection of Salmonella sp. 23S r RN A (Nordento ft , Ch ristensen, and W e gener 1997; Vieira - Pinto et al . 2005, 2008). The specificity o f t he probes w as check ed usin g t he BLAS T database ( Altschul et al. 1990) (at http:// blast.ncbi.nlm.nih.gov/Blast.cgi) . The detection probe w as label ed w ith the DIG Oligonucleotide Tailing Kit (Roche Di agnostics, M annheim, Germany) according to t he manuf acturer’s instructions. 4.2. 8. GENERA TION OF THE IN VITRO 23 S rRNA TRANSCRIP T The target gene w as amplified by PCR using crude cell extract o f S. Typhim urium as a tem plate a nd 10 pmol o f the f orward and reverse p r imers ( Table 4. 2). The forward primer contained at the 5′ – en d the T7 promoter sequence CTA A T A CGA C TC ACT ATA G GG. The P CR conditions were a s follows: 3 min at 95°C; 3 5 cycles of 15 s at 95°C, 30 s at 65,5°C and 1 min 45 s at 72°C; 10 min at 72°C and a f terwards held at 4°C. PCR p roducts were analyz ed by agarose g el electrophoresis an d purified with the QIAquick PCR pu rificat ion k it (Qiagen, Hilden, Germany). Puri f ied PCR pr oducts were quantif ied by absorbance m easurement at 2 60 nm (GeneQuant II, Viochrom Ltd., Cambridge, UK). Target RNA w as generated f rom the puri f ied PC R product by means of T7 RN A polymerase in vitr o transcript ion using the MAXI script™ T 7 - kit (Ambion, Austin, TX, USA). T he qualit y of the in v itro 4. RNA based detection of Salmonella 80 transcribed RNA w as c hecked us ing the Agilent 2 100 Bioanalyser and RN A 6000 Nano LabChip Ki t (Ag ilent T echnolog ies, W aldbr onn, Germany). RNA w as q uantified by absorbance measurement a t 260 nm (NanoDrop ND 1 000, Thermo Fisher Sci enti f ic, USA). Table 4. 1. Seq uences of the oligonuc l eotid e probes used in san dwic h hybri diz ation for the detec tion of S. T yphi murium 23S rRNA ( Taskila et al . 20 11) . Probe nam e Pr obe specifi catio n Probe seq uence 5’ – 3’ Location of probe Probe modification Sal set Sal3_1968 Detection AGGAGC TTCGCT TGCGC TGA 1968 – 1987 3’ - digox igenin Sal3_1816 Capture TAAATCACTTCACCTACGTG 1816 – 1835 5’ - biotin ST set ST 23S_311 D etection TCACC CTGTATCGCG CGCCT 311– 33 0 3’ - digox igenin ST 23S_348 C apture CGAGCTCACA GCACATGCG C 348– 36 7 5’ - biotin ST 23S_291 H elper TT CCAGACGC TTCCACTAA C 291– 310 ST 23S_368 H elper ACGTGTCCCGCCCTAC TCAT 368– 38 7 PCR prim ers ST 23S_T7d90 Forward PCR primer (CTAATACGACTCACTATAGGG) TA TGA AC C G TTA TA AC C GG CG 90 - 110 5’ - T7 promoter ST 23S_rev2068 Rever se PCR primer CATCATTACGCCA TTCGTGC 2068 - 2087 4.2. 9. A N A LYSIS OF TH E 2 3S rRNA A MOUNTS For the detection of the S. T yphimurium 23S rRN A molecules, the fluorescence SHA (Rautio e t al. 2003) was used. SHAs w ere c arried out in 96 - well brig ht U - shaped microplates (Gr einer - Bio - One 4. RNA based detection of Salmonella 81 GmbH, Frickenhausen, Germany) usin g a shaking incubator (Thermomixer Comfort, Eppendorf). A total of 10 μ L of the cru de c ell extract was mix ed with the 5 × saline - sod i um ci tra te bu ff e r (S SC) hybridization buffer (0.75 M sodium chloride, 0.075 M sodium citrate, pH 7.0), 20% demonized formamide, 3% dextran sulphate, 0.2% T W E EN20, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0. 02% bovine serum albumin , 5 p mol biotin - labeled c apture probe, 0 .1 pmol D I G - labeled detection probe , and 3 pmol of each helper probe in a to tal volume of 100 μ L. T he hybridization reactions were performed in 3 parall els at 750 rpm and 55° C fo r 30 m i n. A t ot a l of 1 5 μ L of Strept avidinM ag neSphere® P ar am ag ne t ic Particles (Promega, M adison, W is ., U.S.A.) w as washed as recommended by t he manufacturer, and adde d to each w ell af ter the hy bridization react ion and incubated at 55° C , 750 rpm , for 30 min. Subsequently, beads were w as hed 3 ti mes w ith 120 μ L of a was hing buff er (1% SSC, 0.01% sodium dodecyl sulfate [SDS]) at 25°C, 750 rpm , for 2 min. D uring the w ashing step, bead s were kept in w ells with a M ag naBot® 96 M a gnetic Separation Dev ice (Prom ega). A t otal of 100 μ L o f anti - DIG alkaline phosphatase Fab - fragmen ts (Roche Diagnost ics) were diluted w it h 1 × SS C, 0.1% TW EEN20 t o a final concentration of 375 U/L, and applied to each well. T he 96 - well plate was incubated at 25° C , 500 r pm , for 30 m in. Unbound enz yme was re moved by w ashing 3 times as descr ibed above. Sa m ples were transferred into a new micr o plate and washed once more. Final ly, 100 μ L of 1 mM AttoPhos® f luorescent substrate (Prome ga) was added to each well, and the enzymatic reaction was performed at 37° C, 750 rpm , for 20 m in. Sample portions of 90 μ L we r e trans f erred into a C ostar black 96 - w ell assay plate (Corning Inc., Cornin g, N.Y., U.S.A.) for the f luorescence measurement w ith a W al lac Victor multila bel counter (PerkinElmer Life Sciences, Turku, Finland) at an ex citation wavelength of 450 nm and an emission w avelength of 560 nm. The propo sed sche me for the total an alysis procedure is presented in Fi gur e 4 . 2. 4. RNA based detection of Salmonella 82 RESULTS 4.3. 1. D EVELOPMEN T OF SHA FOR SALMONEL LA TYPHIMURIU M 23 rRN A For the detection o f Salmonella typhimurium in mix ed cultures, two sets o f specific oligonucleotide probes targeting the 23S rRNA w er e designed (Table 4.1, Figure 4.3). The f irst set, Sal, of capture and detection probes is based on t he existing Sal 3 probe used in the FIS H method for detection o f Salmonella sp. 23S r RNA (Nordentoft, Christensen, and W egener 19 97; Viei ra - Pinto et al. 2005, 2008) . The second set, ST, w as de novo designed in this s tudy, and contained tw o helper probes in addition to the capture and det ection probes . Due to the fac t that t he ribosomal RN A is v er y conservative among many bacteria species, finding specific sequences of S. Typhimurium 23r RNA for the probe des ign is very chal lenging . Since it was impossible to get one probe specific only to S. Typhimurium, we designed the probes so, that each detection and cap t ure probes w ere specif i c to differ ent species, and they w ere not int erfering. T h e f r ag m e nt of S. T yphimurium 23r RNA w as sy nthetized in vit ro (Figure 4.4) and u sed a tar get molecule for testing the ST set SHA p robes (Figure 4.5). A concentration of 6 × 10 8 ( 1 f m o l) t arg et molecules in the hybridization solution gave a s ignal that w as signif icantly above the detection limit. The reaction was linear over the tested r ange, w hich was up to 3 × 10 10 (50 f mol) of target molecules . 4. RNA based detection of Salmonella 83 Fig. 4.3. Seq uence of the Salm onella typhim urium 23S ribos om al RN A gene (Ge neBank : CP009 102.1) . The Sal set of the pro bes (Sal3 _1968 and Sal3 _1816) is locat ed cl oser to the end of the gene, and t he ST set (ST23 S_31 1 and ST 23S_348 in combi nation with help er probes S T 23S_2 91 and ST2 3S_368) is close r to the b eginning. 4. RNA based detection of Salmonella 84 Fig. 4.4. In v itro s y nthesiz ed fragm ent of S. Typhi m urium 23r RNA (Agilent 2100 Bioan alys er). The fir st t wo lines are the sam e fragm ents tr anscribed inde pendentl y. The lengt h of th e fragm ent i s 2081 b ases, the conc entrati on of th e RNA i s about 90 ng/µ l. Fig. 4.5 . Standar d curve of the S alm onella typhi m urium 23S rR NA sandwich hybri diz ation ass ay wi th ST s et of the prob es . The error bars s how the ±SD of three parall el exper im ents. 4. RNA based detection of Salmonella 85 4.3. 2. DETECTION O F SALMO N ELLA TYPHIM URIUM IN ME A T S AMPLES USING S HA In order to test the applicabil ity of the designed probes, Salmonella cells were inoculated in RVS medium and samples for the SH A were collected af t er 2. 5 a nd 5 . 5 h of c u lt iva t i on . T he sp e c if i cit y of the SHA was i ncreased by preparing the hybridization reaction at 55° C instead o f the com monly used 50° C ( R a ut io et al. 2003). The combination of probes Sal3_1968 (d etect ion) and Sal3_1816 (capture) resulted in f luorescent si gnals just slightly abov e t he assay bac kg round (Figure 4.6). The signals obt ained by using the probe set ST23S _311 (detection) and ST23S_348 (capture) in combination with helper probes S T23S_291 and ST23S_368 were significantly higher than the background, and thereby, this set w as used in f ur ther experim ents. The specificity of the SHA w as test ed by analyz ing 9 minced meat samples incubated f or 1 8 h in B P W (F ig ur e 4 . 6) . St a t i s t ic a ll y, the differ ence be t ween si g nals from the meat and t he assay background w er e not significant ( P = 0.8 ), showing that the developed SH A is spe cif ic f or the de tection of Salmone lla 23S rRNA in minc ed meat. In order t o ensu re the accuracy of the SHA, t he signals from minced meat samples (individually prepared for each experiment) w ere used as the assay background in further studies. Fig. 4.6. Sa ndwich hybr idizat ion assay for S. Typhim urium 23S rRNA. White bar s = SHA with usin g Sal s et of probes; black bar s = SH A with us in g the ST probes design ed in thi s resear ch. 1 and 2 = fluor escence s ignals for S. Typhim urium sa m ples coll ecte d aft er 2.5 and 5.5 h of incuba tion in RVS m edium, respect i vely. Gray bars = backgr ound si gnals. AB = pure ass ay bac kgro und; MB = m eat backgr ound. Err or bars sho w the SD of 3 paral le l m easurem ents (T askila et al. 2011) . 4. RNA based detection of Salmonella 86 The effect o f the modi f ied conditions on t he recovery o f heat i njur ed S. t yphimurium in mix ed cultures was studied by a cult ivation o f spiked minced meat sa mples. The 23S rRN A q uantification by means of SHA w as used for the com parison. In o r der to show the efficiency o f the r ecovery w it h respect to the surviv al in select ive medium, minced meat samples and approximately 60 CFU o f heat - i nj ured Salmonella cells were first incubated for 4 or 6 h i n common or modi f ied BP W and then i n selective RVS medium. The minced meat samples without spiked Salmonella cells were cultivated under the same conditions and us ed as negative controls. S amples for 23S rRNA quantificat ion were take n from the RVS cultures a f t er 8 h of incubation. A fter 4 h of incubation, there was no statistically significant difference betw een the tes ted conditions ( P = 0.33) (Figure 4.7). Based on the results, t he concentr ations of Sal m onell a cell s in both media hav e been relatively low, indicating that 4 h of incubation in nonselective medium was not lon g enough for sufficient recovery in either conditions. This supports the results by (Zhao and Doy le 2001), who claimed that an incubation time o f less tha n 6 h w ould not assure recovery of heat - i nj ure d Salmonella cells. After 6 h of non selective cultivation however, the di ffer ence between the 2 condition s was statistically signif icant ( P < 0.05), showing that the use of modi f ied conditions improved the recovery in comparison to the common BP W . 4. RNA based detection of Salmonella 87 Fig. 4.7. C om paris on of comm on (white bars) and modi fied BPW ( bl ack bars) for r ecover y of heat - injur ed S. Typhi muri u m in different minc ed m eat sam ples (1 to 4). T he fig ure presents fl uorescent si gnal to backgr ound r atios m easured wit h the SHA for enric hm ent cultur e s of 4 diff erent meat sam ples, incu bated for 4 or 6 h in BP W. Er ror b ars sh ow t he SD of 3 par allel m easurem ents (Task ila et al. 2011 ) . DISCUSSION In this wor k, we present r esults which show t hat th e developed Salmonella - specif ic 23 S rRNA SHA is a potential tool f or rapid detection o f Salmonella in foods even i n the presence of hi g h concentr ation o f background flora. Given the wi de r ange o f sources from which samples could be test ed, hi gh concentr ations of background flora are to be ex pected, and devel oping a met hod that is still specif ic in those circ umst ances is challenging but essential. Although w e addres sed this wide 4. RNA based detection of Salmonella 88 range of sources by ch oosing four di f f erent meat samples, further studies w il l be required for expanding the range of types of samples that can be analyzed w ith t he method and v alidat ing it for routine use. Besides selectivity, another increas ing important re quirem ent is the speed o f det ection. Salmonella typhimur ium is becoming increasingly diff icult to treat wi th conventional antibiotics. T he scale of the antibiotic res istance problem is f urther illustrated by the W orld Health Organization list that was published in 2017 . One of the consequences of increas ed an t ibiotic resistance, is that intensi ve charact eriz ation of an inf ectious microbe is required, to simply identify antibiotics that are still effective (a problem that w ill only increase as more M DR str ains ar e encountered). This charact eriz ation will also lead to longer hospital ization and significantly increased costs. To this extent, m ethods that can rapidly and precisely detect Salmonella contamination in early stages, especially in high flora b ackground, are increasingly val ued. W e describe an optimiz ation of the conditions of recovery culti vation when compared to the common B PW r ec overy. This resulted in a strong, statistically signif icant signal after onl y 6 hours o f recovery (Figure 4.7). W hen c omp ared to the co mm on cultiv ation met hod, a significant increase in spe cif icity of the fluorescent signal could be det ected relative to the bac k ground in all meat sa m ples tested. 5. Summary of results and outlook 89 SUMM A R Y O F RES ULTS AND OUTLO OK Our increased understanding of (and the large avai labilit y of data on) nucleic acid and protein structur es together w ith seq uences, has gone hand i n hand w ith ( and ena bled) the development o f highly specific detection methods targeting small parts of the structur e or sequence. T hese methods have become an e ssential tool in health di agnostics, to s creen for dr ug efficacy, to anal yze contaminants in food an d water s amples, t o analyz e environment al pollu tion and even to monitor recombinant production. But, as hi gher standards and requirements are continually bein g implemented in terms of health diagnostics an d food safety, so do the re quir ements for their detect ion speed and accuracy continually increase. In this thesis, we hav e developed , teste d and validated sandwich hybridization technology for th e detect ion and q uantification of recombinant pro tein and nucleic acids lev els in a variety o f crude cell ex tracts . S A N DWI CH HYBR IDI Z A TI ON FO R PROTEIN DE TECTION In this wor k, we devel oped and demonstrated t he applicability of the sandw ich hybridiz ation (or sandwich ELISA) f or the quantif ication of the mul timer ic (consistin g of di ffer ent subunits ) re combinant collagen pro ly l -4- hydroxylase tetramer protein in cr ude samples. The specificity of both the antibodies used in the assay against the di ff erent collagen prolyl -4- hy dr oxylase subunits , ensures detection of only t he active holoenzyme, not its separate subunits. T hese inactive subunits (which are used as tar gets in commercially av ai lable CP4H ELI SA ) are produced at si gnif icantly differ ent ra tios, and, w hen taken as a target for de tection, can g ive w idel y inaccurat e val ues of ov er all active CP4H production . The de tection of only the assembled CP4H g uarantees the st raig ht correlat ion w ith t he p r otein activity, whi ch has b een proven in this study. This ma k es t he metho d unique among the other available methods f or t he CP4H quantification. Usin g an experimental design approach the antibody concent rations in t he ELISA were optimiz ed. This resulted in th e s uccessful detect ion of recombinant CP4H quantities in cr ude cell extracts of E. coli and could be applied to continually monitor /optimize production lev els during recombinant expression. 5. Summary of results and outlook 90 S A N DWI CH HYBR IDI Z A TI ON FO R SPORE DETE CTION In this study, a p r ocedure w as developed f or detecting Bacillus subtilis spores by utiliz ing an RNA based sandwich hy br idization assay. The procedure included spore activation, germination, and enrichment cultivation, cell di srupt ion, and RNA analy sis using sandwich hybridizatio n assay. T he method showed no s ensitiv it y limitations as it is adap table by en richment cultivation time. T he detect ion procedu re is e asy to per form since the crude cell extract can be used direc tly f or analysis with SHA and no additional RNA purification is required. I n addition to the m et hod being quantitative, it was possible to es t imate the a m ount of 16S r RNA molecules per cell at different stages o f growth. S A N DWI CH HYBRI DIZ A TION FO R FOOD P A THO GEN DETECTI ON W e showed the potential o f RNA based S HA for the detection of m icrobi al pathogens in food sam ples, s pecific ally with Salm onella . Here , an ac curate and rapid detection m ethod w as developed , alongside with an i mproved method f or the r ecovery and en r ichment cultivation, to detect heat injured bacteria o rig inat ing f r om a variety of meat sa m ples. In most detection assay s , a main difficulty f or the accurate detect ion is the presence o f a diverse background flora in the meat samples . Using t he SHA prob es designed f or 23S rRNA of S. t y phimurium , we w ere able t o speci f ically detect Salmonella wit hout interference f r om t he background f lor a, which is hig hly abundant in the sa mples. This method also enabled a more rapid recovery o f heat injured S almonella , speeding up th e ov er all time of detection. A DV A NT AGES OF SANDWICH HYBR I DIZ A TIO N TECHNOLOG Y The main advantages of this technology include the high selectivi ty to wards a target, possibili ty of adapting the system to d etect various targets in a variety of diff erent envi ronment s and the potential to be used w ith different read - out systems and in high throughput assays. The high select ivity of the method is achieved by using t wo molecules spe cif ic to di ffer ent regions of a target for capture and detection. The detection molecule binds to an oth er part of t he target t han 5. Summary of results and outlook 91 the capture molecule , result ing in a “sandwich” structur e. In case of pr otein detection, these molecules are antibodies for dif ferent epitopes of the pr otein. The addi tional advantage of this approach is that m ultimeric proteins, containing two or more subunits can be detected as a whol e complex, when all the subunits are assembled together. This is achieved by designing the antibodies against different subunits o f the protein. The capture and de tection oli gonucleot ide probes ensur e the selectiv ity of the assay for nucleic acid detect ion. Just one or two nucleotide m ismatches in the probes compare d t o the targ et would lead to a dramatic signal drop. Therefore, the selective probes can be designed specific ally f or almost any RNA or DNA t arg e t , even t o d isting uish close related organisms. The method has pre vio u s ly been proven to be e ff i cient for various biological samples, such as water (Leskela et al. 2005), brew er y y east slurr ies (Huhtamella et al. 2007), minced meat ( T askila et al. 2011 ) , bacterial cultures (Neubauer et al. 2007; Soini et al. 2008; Thieme et al. 2008; Rautio et al. 2003) , without the need for t arget purificat ion. Besides used in the present work a f luorescent meter, di ff erent read - out systems can be applied f or detect ing g enerated signals, such as chip - bas ed fluorescent biosensors (W ang et al. 2013 ) or electrical biochip read ers ( Jurgen et al . 2005; E lsholz et al. 2006; Pioch , Schweder, and Jurgen 2008; Pioch et al. 2008; Gabig Cimins ka et al. 2004) . The SHA coupled w it h capillary electrophor esis called TRAC (transcript analysis with aid of affinity capture) w as developed f or multiplex transcript analys is (Rautio et al. 20 06, 2008) (PlexPress Oy, Finland). DETECTI ON T A R GET AND SA M PLE DI VE RSIFI CATION An important question related to the devel oped sandwich hy br idizatio n technology to detect multimeric proteins and nucleic acids in this t hesis, is the transfer o f knowledge to iden t ify new bio - molecular t argets. The seque nce diversity between or ganisms, and the recent la rg e availabili t y of data (t hrough for instance high - throughput sequencing e f forts ), hav e enabled us t o desi g n these targets with great accuracy . T he high speci f icit y o f sandwich hy br idization detection allo ws t h is method to ev en dist inguish be t ween v er y closely related species, or homo logous proteins (as l ong as the sequences are no t 100% identical). 5. Summary of results and outlook 92 For instance, the sandwich ELISA m ethod to detect multimeric proteins, althoug h only validated f or the det ection of active CP4H product ion, could serve as a potential guide for the detection of multimeric proteins in a variety o f c linical and food sam ples. O f particular interest could be the formation of amy loid protein aggregates that for m in many degenera tive diseases, such a s Alzheimer’s disease and Parkinson’s disease. In all these diseases, pro tein misfolding and aggregation result in dy sfunct ional versions tha t can deposi t around cells and in f luence the function of tissues. The ability to rapidly detec t misfolding and a gg r egation events in pr oteins could h elp early diagnosis, which could f a cilitat e in a timely t reatment. The developed here procedure for B. subt ilis spore detection could also b e applied to detect other spore - forming bacteria, of w hich several species f orm health threats. Sinc e germinat ion appears to be triggered w ith quite general and identical s t imuli, the method can easily be adapted to di f ferent organisms, as long as the g enomic sequence is a vailable t o design specific and uni que prob e s. For the speci f ic de t ection of Salmonella, testing of its speci f i city in a variety o f other food and w at er sources is an i nteresting follow - up di rect ion. As previously discussed, Salmonella c an inf ect through a wide range of food and water sources, and can do so in a w ide variet y of climat es. And even though Salmonella infections have been studied f or a lon g t ime, the spread o f the bacteria, and track ing the source o f outbreaks, o ft en re mains extremely di ff icult. This is in large part due t o diff i c ultie s in corr ect iden tificat ion of the disease in complex bi ological envi ronments. T her ef ore , the ability to identif y the pres ence of Salmonella in a wide variety of samples, allows better ident ification of the spread of outbreaks, and hence could pr ov ide key insight in prev ention of future outbreaks. How ever, prior to i mplementation, the correct identi f i cation of Salmonella cells us i ng S HA b as ed detect ion methods in a wide r r a ng e of sour ces should be properly tested and v alidated. Another interest ing possible adaptation from SHA detection in Salmonella , is the detection of other antibiotic res istant bac teria. Antibiotic resistance is a rapidly growi ng problem and treatment i s becoming increasingly diff icult. This means that in more and more cases, correct identificat ion of the bacterium is requir ed, before effective treatment can be started. So, adapti ng SHA for the rapid and accurat e detection o f the bacter ia could i ncreas e e f f ectiveness of treatments, but also significantly reduce the costs for hos pitalization. 5. Summary of results and outlook 93 HIGH THRO UGHPUT S CREENI NG & AUTOM ATION Finally, there is the q uestion o f the development o f scale up methods and a nalysis. A decisive f act or in the applicabili ty of de tect ion me thods, relates to the cost o f individual detection reaction and possibility of scaling. And when considering the targets of our dev eloped met hods, lar ge sample numbers are to be exp ected. The detection of multimeric proteins was designed to continually measure production over long time - scales , resulting in m any sa mpling events . B o t h s por e - f or m ing bacteria and Salmonella can po tentially pose serious health problems, c an be present in a w ide var ie t y of environments and in f ec tions can spread with gr eat speed. This also illustrates the need f or f r eq u e nt testi ng across a wide array of sources, resulting also in many samples . 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