The development of a competitive PCR-ELISA for the detection of equine herpesvirus-1

The de elopmen o a compe i i e PCR/ELISA o he de ec ion o equine he pes i us-1 P. Daly, S. Doyle  Na ional Ins i u e o Cellula Bio echnology, Depa men o Biology, Na ional Uni e si y o I eland, Maynoo h Co., Kilda e, I eland Recei ed 12 June 2002; ecei ed in e ised o m 10 Oc obe 2002; accep ed 11 Oc obe 2002 Abs ac Equine he pes i us-1 (EHV-1) in ec ion is o signi ican animal wel a e and economic impo ance. Ye , no s anda dised molecula echniques a e a ailable o diagnosis o con i ma ion o i al in ec ion. The pu pose o his s udy was o de elop a s anda dised and quan i a i e assay sys em o he eliable de ec ion o EHV-1 in ec ion which was capable o elimina ing he likelihood o alse nega i e esul s. A egion wi hin he EHV-1 glycop o ein B gene was ampli ied by polyme ase chain eac ion (PCR), cloned and subjec ed o si e-di ec ed mu agenesis o gene a e a con ol plasmid, ampli iable by iden ical p ime s o wild ype EHV-1, ye capable o de ec ion by an al e na e dini ophenyla ed oligonucleo ide p obe in a PCR/ELISA sys em. A compe i i e PCR/ELISA sys em which can con ol o he p esence o PCR inhibi o s and which is capable o de ec ing 63 genome equi alen s o EHV-1 has been de eloped. EHV-1 p esence in in ec ed equine issue and cell cul u e ma e ial was demons a ed using his sys em. The en i e assay can be comple ed wi hin one wo king day and acili a es mul iple sample analysis. The a ailabili y o a obus , compe i i e PCR/ELISA sys em o he de ec ion o EHV-1 will acili a e he apid and sensi i e de ec ion o EHV-1 and o e s he po en ial o elimina ing he occu ence o abo ion s o ms in s ud a ms. #2002 Else ie Science B.V. All igh s ese ed. Keywo ds: Fe al; Diagnosis; Oligonucleo ides; Immunoassay; Molecula diagnos ics 1. In oduc ion The alphahe pes i us, equine he pes i us-1 (EHV-1), is he causa i e agen o espi a o y disease, abo ion and neu ological diso de s in ho ses (Allen and B yans 1986; C abb and S udde , 1995). O iginally EHV-1 was designa ed as hinopneumoni is i us, howe e exam- ina ion o es ic ion endonuclease DNA inge p in s by S udde e al. (1981) showed wo dis inc i us sub ypes namely EHV-1 and EHV-4. While EHV-4 is es ic ed o he espi a o y epi helium and lymph nodes d aining he lung, EHV-1 is dis inc in ha i sp eads sys emically esul ing in pos - espi a o y complica ions including abo ion and pa alysis (Wagne e al., 1992; Law ence e al., 1994). Due o he abili y o EHV-1 o cause ‘abo ion s o ms’ in ma es a e e al in ec ion (S udde and Blackney 1979; C abb and S udde , 1995) diag- nosis o EHV-1 mus be apid and sensi i e so ha ea ly in e en ion policies, aimed a educing he e ec o i us sp ead, can be pu in place (Ballagi-Po dany e al., 1990; Sha ma e al., 1992; Law ence e al., 1994; Gup a e al., 1996). The de elopmen o he polyme ase chain eac ion (PCR) has lead o imp o ed de ec ion o EHV-1 DNA om clinical and cell cul u e ma e ial (Ki isawa e al., 1993). Ampli ica ion o i al DNA by PCR can be u he enhanced by hyb idisa ion wi h i al speci ic DNA p obes (Law ence e al., 1994; Welch e al., 1992; Ki isawa e al., 1993) and also by es ic ion endonu- clease diges ion (Allen e al., 1983; Mo is and Field, 1988; Pal i and Ch is ensen, 1995). Howe e , he ex ac ion o i al DNA om samples may also lead o he co-pu i ica ion o PCR inhibi o s (Ze bini e al., 1999). The p esence o inhibi o y molecules in a PCR eac ion supp ess Taq polyme ase ac i i y, p e en ing he ampli ica ion o i al DNA and hus leads o alse nega i e esul s (Ba be e al., 1999). A me hod o de e mining he p esence o PCR inhibi o s is co- Co esponding au ho . Tel.: /353-1-7083858; ax: /353-1- 7083845 E-mail add ess: [email p o ec ed] (S. Doyle). Jou nal o Vi ological Me hods 107 (2003) 237 /244 www.else ie .com/loca e/j i ome 0166-0934/02/$ - see on ma e #2002 Else ie Science B.V. All igh s ese ed. PII: S 0 1 6 6 - 0 9 3 4 ( 0 2 ) 0 0 2 5 2 - 5 ampli ica ion o a con ol plasmid wi h he a ge DNA using iden ical p ime s pai s (Ba be e al., 1999; Ze bini e al., 1999; D ews e al., 2000). The con ol plasmid con ains a sho mu agenised DNA sequence and amplicons o a ge and con ol can be dis inguished om each o he , pos -PCR, using ype-speci ic oligonu- cleo ide p obes in an ELISA. In he p esen s udy, he de elopmen o a obus compe i i e PCR/ELISA sys em o he de ec ion o EHV-1 is desc ibed. The con ol plasmid was con- s uc ed by he in oduc ion o a he e ogeneous dini- ophenol (DNP)-labelled oligonucleo ide p obe sequence by si e-di ec ed mu agenesis (SDM). The apid, compe i i e PCR/ELISA assay displayed high le els o sensi i i y and speci ici y de ec ing as li le as 63 genome equi alen s o EHV-1. 2. Ma e ials and me hods 2.1. EHV-1 i al s ains and i al DNA ex ac ion EHV-1 wild ype (w ) s ain Ab4p (ob ained om D Josh Sla e , Queens Ve e ina y School Hospi al, Uni- e si y o Camb idge, UK) was o iginally isola ed om a ield case o abo ion and pa alysis in a ma e and is ega ded as a pa hogenic s ain o EHV-1. The i us was supplied as a concen a ed mix u e o i us in lysed ED cells wi h a i al copy numbe (genome equi alen ) o app oxima ely 10 10 , as de e mined by plaque assay. EHV-1 issue homogena es (p o ided by D We ne Eichho n, Mu ¨nchen, Ge many) we e isola ed om e al li e , lung and spleen. Posi i e EHV-1 issue samples (by IF and isola ion) we e labelled S726-8, S685-7 and 835-7 and nega i e issue samples labelled S611-3, S723-5 and 597-9. EHV-1 w s ain Ab4p (5 ml lysa e) and issue homogena e (5 ml) we e ex ac ed using he Qiagen QIAamp spin column DNA midi ki ex ac ion p o ocol (Qiagen, Hilden, Ge many) ( inal olume: 200 ml). DNA ex ac ion was pe o med in sepa a e a ea o ha o PCR eagen p epa a ion and nucleic acid ampli ica ion. 2.2. EHV-1 oligonucleo ide p ime s The i al DNA was ampli ied by using speci ic oligonucleo ide p ime s selec ed om he glycop o ein B (gB) egion o he EHV-1 genome. The p ime sequences we e selec ed based on esul s ob ained by Ki isawa e al. (1993). The 20 base bio inyla ed (B) o wa d p ime sequence (P1) was compa ible wi h bo h EHV-1 and EHV-4 while he e e se 20 base sequence (P2) was compa ible wi h only EHV-1. The 5?bio iny- la ed nucleo ide sequences o he o wa d p ime P1 was: 5?-B-CTTGTGAGATCTAACCGCAC-3?and un- modi ied e e se P2 p ime was: 5?-GCGTTATAGC- TATCACGTCC-3?(Sigma-Genosys, Camb idge, UK). 2.3. Ampli ica ion o DNA A o al o 5 ml o each DNA sample was used in each PCR eac ion wi h 10 mM T is/HCl pH 9.0, 50 mM KCl, 0.1% ( / ) T i on X-100, 1.5 mM MgCl 2 , 200 mM o each dNTP (P omega, WI, USA), 1 M Be aine (Sigma, Do se , UK), 1.0 mM o he abo e p ime s in a o al olume o 49.5 ml. Ho -s a PCR was pe o med on each sample by adding 0.5 mlo Taq polyme ase (P omega) a e he ini ial 6 min dena u a ion s ep a 95 8C. The PCR eac ion was hen con inued wi h 35 cycles consis ing o 1 min dena u a ion a 95 8C, p ime annealing a 55 8C o 1 min, ex ension a 72 8C o 2 min and inally ex ension o 5 min a 72 8C. PCR ampli ied DNA was analysed by aga ose gel elec o- pho esis whe eby 10 ml o p oduc was an on 1% (w/ ) aga ose (P omega) con aining 0.5 mg/ml o e hidium b omide (Sigma, Do se ) o 30 min a 100 V. Visualisa- ion o he 460 bp P1/P2 amplicons was pe o med using an ‘Eagle-Eye II’ digi al s ill ideo sys em (S a- agene, CA, USA). 2.4. Cons uc ion o EHV-1 gB plasmid EHV-1 gB P1/P2 amplicons we e pu i ied using he S a a P ep PCR pu i ica ion ki (S a agene). Pu i ied PCR p oduc s we e cloned in o he pCR-2.1 TOPO ec o (In i ogen, G oningen, NL) ollowing manu ac- u e s’ ins uc ions and ans o med in o compe en E. coli. Following pu i ica ion (Qiagen plasmid pu i ica- ion ki , Qiagen), he gB plasmid was subjec ed o PCR using plasmid-speci ic M13 p ime s supplied by he manu ac u e and p oduc s sc eened by es ic ion diges ion wi h EcoRI and BamHI enzymes. The M13 PCR p oduc s we e sequenced using a Pe kin Elme ABI P ism 310 gene ic analyse and sequence homology was de e mined wi h ha o he known w EHV-1 gB sequence using he BLAST algo i hm (h p:// www.ncbi.nlm.nih.go /blas /bl2seq/bl2.h ml). 2.5. Cons uc ion o compe i i e in e nal con ol plasmid A modi ica ion o he me hod o Ba be e al. (1999) o SDM was employed in he design o he con ol plasmid which in ol ed eplacing a 30 bp sequence o he gB plasmid wi h a he e ogenous sequence o simila T m and deoxynucleo ide composi ion (Fig. 1). The o wa d 40-me p ime (mu an DNP) con ained he con ol p obe si e. The i s 10 bases a he 3?end o he ‘mu an DNP’ p ime was complemen a y o he EHV-1 gB sequence while he emaining 30 bases o med a 5? o e hang which inco po a ed he con ol p obe si e (Fig. 1). The sequence o he mu an DNP p ime was: 5?-CTCAAGAAGACc gcagTC c aga GGAACTCCAA- CGTCA-3?(no e: he con ol p obe si e in bold). Two es ic ion si es, Ps IandXbaI we e inco po a ed in o P. Daly, S. Doyle / Jou nal o Vi ological Me hods 107 (2003) 237/244238 he con ol p obe sequence, which allowed o selec ion o con ol plasmid (shown in small on ). The 20-me ‘ e e se back/back’ p ime shown below was phos- pho yla ed a he 5?end (5?-þ-CCTCCACTGCTC- GTTTGTGT-3?). In e se PCR was ca ied ou using 2.5 U o P u polyme ase, in a o al olume o 50 ml wi h 1.0 mM o each o he abo e o wa d ‘mu an DNP’ and ‘ e e se back/back’ p ime , 20 mM T is/HCl pH 8.8, 10 mM KCl, 10 mM (NH4) 2 SO 4 , 2 mM MgSO 4 , 0.1% ( / ) T i on X-100, 5 mg BSA, 1.5 mM MgCl 2 , 200 mM o each dNTP and 1 ml empla e (gB plasmid: 70 mg/ml). Cycle condi ions we e ini ial 4 min dena u a ion s ep a 95 8C. The PCR eac ion was hen con inued wi h 20 cycles consis ing o 1 min dena u a ion a 95 8C, p ime annealing a 60 8C o 2 min, ex ension a 72 8C o 7 min. Ten mic oli e o PCR p oduc was emo ed and gB plasmid diges ed o 30 min a 37 8C wi h DpnI (10 U) in SuRE/Cu bu e (33 mM T is ace a e, 66 mM po assium ace a e, 10 mM magnesium ace a e, 0.5 mM DTT, pH 7.9). The linea con ol plasmid was hen liga ed and cloned in o one-sho compe en E. coli and colonies selec ed o he p esence o he plasmid by PCR using he P1/P2 p ime s pai s. The con ol p obe- speci ic es ic ion si es (XbaIandPs I) we e subse- quen ly used o sc een and selec P1/P2 posi i e colonies o he co ec con ol p obe si e sequence. Pu i ied gB and con ol plasmids we e dilu ed 1/100 in s e ile wa e (DNA/RNA ee) and he O.D. 260/280 nm measu ed using a Beckman DU 640 Spec opho ome e . O.D. 260/dilu ion ac o /50/concen a ion o DNA in mg/ml. 2.6. PCR/ELISA p ocedu e Mic owells (Nunc, Roskilde, Denma k) we e coa ed wi h s ep a idin (2.5 mg/ml) and s abilised o 4 8C s o age by he addi ion o 1% (w/ ) BSA in 50 mM sodium ca bona e bu e pH 9.4. P1/P2 PCR p oduc s (5 ml) we e ans e ed in duplica e o s ep a idin coa ed mic o i e pla es in 95 ml 6X SSC (0.9 M NaCl, 0.09 M sodium ci a e solu ion). The bio inyla ed amplicons we e le o a ach o 30 min a 37 8C. Wells we e washed wice wi h phospha e bu e ed saline con aining 0.05% ( / ) Tween-20 (PBST) and 100 mlo mel ing solu ion (0.125 M NaOH, 0.1 M NaCl) was added o he mic owells o dissocia e DNA duplex, incuba ed a oom empe a u e o 3 min and washed ou imes wi h PBST. Then, 100 ml o ei he he gB o Fig. 1. In e se PCR me hod o SDM showing he indi idual s eps in ol ed in he gene a ion o a con ol plasmid. ‘Mu an DNP’ e e s o an oligonucleo ide con aining a complemen a y sequence o which he con ol DNP p obe binds. P. Daly, S. Doyle / Jou nal o Vi ological Me hods 107 (2003) 237/244 239 he con ol complemen a y DNP oligonucleo ide p obe (100 ng/ml) in 6X SSC/0.1% (w/ ) SDS was hyb idised o he bio inyla ed o wa d s and a 60 8C o 1 h ollowed by washing ou imes wi h PBST. The DNP oligonucleo ide p obe was designed o be complemen- a y o he gB o con ol sequences desc ibed abo e. The DNP p obes (OSWEL, Sou hamp on, UK) nucleo ide sequences we e: gB p obe: 5?(DNP) 3 -TGACGTTGG- AAGAATCGGTACCTGCCATTG-3?; con ol p obe: 5?(DNP) 3 -GTTCCTCTAGAGACTGCAGGTCTTCT- TGAG-3?. A e hyb idisa ion o he DNP p obes wells we e washed ou imes and blocked o 10 min wi h 2.5% (w/ ) milk powde in PBST. Following emo al o he blocking solu ion, IgG[an i-DNP]-ho se adish pe - oxidase conjuga e was added, incuba ed a oom empe a u e o 30 min and washed ou imes wi h PBST. Subs a e (100 ml e ame hylbenzidine) was hen added and incuba ed o 15 min a oom empe a u e. The eac ion was e mina ed by he addi ion o 1 N sulphu ic acid and measu ed spec opho ome ically a 450/630 nm (Dyna ech MRX). 2.7. Compe i i e PCR/ELISA assay P elimina y expe imen s in ol ed ampli ica ion o he P1/P2 agmen s om se ial dilu ions o gB and con ol empla es in non-compe i i e assays o de e mine op i- mal copy numbe de ec ion. Assay sensi i i y was de e mined using di e en p obe associa ions (i.e., gB p obe alone, gB and con ol p obe oge he and con ol p obe alone). Following op imisa ion o PCR/ELISA condi ions, se ial dilu ions o gB plasmid we e co- ampli ied wi h known di e en amoun s o con ol (da a no shown). The selec ed concen a ion o con ol plasmid used in he compe i i e PCR/ELISA was es ablished by de e mining op imal le els o gB a ge DNA de ec ion whils no supp essing he de ec ion o he con ol. Following de e mina ion o he op imal con ol plasmid copy numbe in he compe i i e PCR/ ELISA assay, alida ion o he assay in ol ed co- ampli ying 1000 copies o con ol wi h pu i ied w Ab4p EHV-1 and posi i e/nega i e EHV-1 e al li e lung and spleen issue samples. Finally, a known PCR inhibi o (sodium iodide) was used o assess he e icacy o he compe i i e con ol plasmid in he PCR/ELISA. 3. Resul s 3.1. Gene a ion o gB and in e nal con ol plasmids The EHV-1 P1/P2 PCR p oduc (460 bp), de i ed om w EHV-1 s ain Ab4p, was pu i ied and cloned in o he pCR-2.1 ec o and compe en one-sho E. coli we e subsequen ly ans o med. Resul an M13 o - wa d- e e se amplicons we e o he co ec size (661 bp) and subsequen diges ion wi h BamHI and EcoRI yielded 613 and 478 bp p oduc s, espec i ely (da a no shown). Sequence analysis o he pu i ied gB plasmid e ealed he co ec o ien a ion o he P1/P2 agmen in he pCR-2.1 TOPO ec o . PCR and es ic ion analysis o he plasmid de i ed om SDM o he gB plasmid esul ed in he gene a ion o he co ec P1/P2 amplicon size ha was suscep ible o diges ion wi h bo h XbaIandPs I(Fig. 2), was hus selec ed as he in e nal con ol plasmid o PCR/ELISA op imisa ion. 3.2. Compe i i e PCR/ELISA Se ial dilu ions o he gB plasmid om 10 2 /10 4 copies we e ampli ied in he absence, and p esence, o dilu ions o con ol plasmid o de e mine he minimum copy numbe o compe i o equi ed o de ec ion by PCR/ ELISA whils no inhibi ing he ampli ica ion e iciency o he a ge . A minimum o 100 molecules o ei he plasmid was equi ed o eliable de ec ion (Fig. 3 and da a no shown). Fu he mo e, compa able ampli ica- ion e iciency was eco ded be ween he a ge and he con ol a simila copy numbe s. Lowe copy numbe s o each plasmid ailed o esul in any de ec able P1/P2 amplicons by ei he PCR/ELISA o gel elec opho esis (Fig. 3 and da a no shown). A o al o 10 000 copies o he con ol when co-ampli ied wi h se ial dilu ions o gB plasmid caused a educ ion in he sensi i i y o de ec ion as de e mined by PCR/ELISA when compa ed o ha o gB ampli ied alone (Fig. 3). In con as , when 1000 copies o con ol was co-ampli ied wi h gB plasmid, no signi ican di e ence in ampli ica ion e iciency o a ge gB in he compe i i e eac ion o when gB was ampli ied alone could be de ec ed (Fig. 3). A 1000 copies o con ol, as low as 100 copies o gB plasmid we e eadily de ec ed in he compe i i e assay and his le el o con ol was subsequen ly used in he compe i i e PCR/ELISA. No signi ican c oss- eac i i y was ob- se ed be ween gB/con ol DNP p obe and he gB/ con ol PCR p oduc s as de e mined by hyb idisa ion o non-homologous p obes wi h amplicons (Fig. 3 and da a no shown). 3.3. Compe i i e PCR/ELISA assay o he de ec ion o w EHV-1 To e alua e he e iciency o he compe i i e PCR/ ELISA assay, 1000 copies o con ol we e co-ampli ied wi h se ial dilu ions o ex ac ed w EHV-1, and issue samples. All samples we e ex ac ed using Qiagen spin columns and subsequen ly se ially dilu ed o 1/10 6 . Each dilu ion was ampli ied alone o co-ampli ied wi h 1000 copies o con ol plasmid. Ampli ica ion o in- di idual w EHV-1 se ial dilu ions ( om a i al s ock o 10 10 genome equi alen s EHV-1) esul ed in endpoin de ec ion a a 10 5 dilu ion, which equa es o 63 P. Daly, S. Doyle / Jou nal o Vi ological Me hods 107 (2003) 237/244240 genome equi alen s o EHV-1 (Fig. 4). In he compe i- i e assay, con ol ampli ica ion was ou -compe ed by he la ge amoun s o w EHV-1 in he ange om nea o 10 3 dilu ion. F om 10 4 o 10 6 w dilu ions, he le els o con ol ampli ica ion inc eased wi h dec easing w concen a ion. Due o ampli ica ion o w EHV-1 a high concen a ions and con ol a low a ge le els, hese esul s demons a e ha no PCR inhibi o s we e p esen in any o he se ial dilu ions es ed. 3.4. Valida ion o he PCR/ELISA The compe i i e PCR/ELISA was alida ed by examina ion o EHV-1 posi i e and nega i e e al issue homogena e samples isola ed om e al li e , lung and spleen. Samples labelled S726-8, S685-7 and 835-7 we e posi i e by PCR/ELISA while samples S611-3, S723-5 and 597-9 we e nega i e(Fig. 5). The esul s ob ained he e co ela ed wi h gel elec opho esis analysis (da a no shown) and immuno luo escen assay/ i us isola ion s udies by D We ne Eichho n (pe sonal communica- ion). Co-ampli ica ion o 1000 copies o con ol wi h ex ac ed nega i e EHV-1 issue samples (S611-3, S723- 5 and 597-9) esul ed in he de ec ion o he con ol amplicon in all h ee nega i e samples (Fig. 5). In he posi i e issue samples, con ol ampli ica ion was no obse ed due o he high empla e EHV-1 le els which ou -compe ed he con ol o P1/P2 p ime binding. A a concen a ion o 50 mM NaI, ampli ica ion was comple ely inhibi ed while a 5 mM NaI, he inhibi o y e ec was almos comple ely emo ed wi h abso bance alues app oaching ha o con ol only (Fig. 5). 4. Discussion In his epo we desc ibe a apid and sensi i e compe i i e PCR/ELISA me hod which can de ec 63 genome equi alen s o EHV-1 om cul u e and clinical specimens. To da e, only a limi ed numbe o epo s de ail he ela i e sensi i i ies o he oligonucleo ide p ime s in EHV-1 PCR de ec ion assays whe eby he sensi i i ies o he PCR assays using ‘ho -s a ’ PCR (Os e iede e al., 1994) o nes ed PCR (Ki isawa e al. 1993; Bo che s and Sla e , 1993) using e hidium b omide/aga ose gel elec- opho esis de ec ion was 100, 10 copies and 1/3 copies o EHV-1, espec i ely. In addi ion, a majo complica- ion in he diagnosis o i al in ec ions by PCR is he p esence o Taq polyme ase inhibi o s ha lead o alse nega i e esul s (Cou ney e al., 1999; Ba be e al., 1999). The p esence o such inhibi o s can be moni o ed by he inco po a ion o a compe i i e con ol plasmid (Cou ney e al., 1999). Thus, bo h he a ge and con ol a e a ec ed equally by a ia ion in ampli ica ion e iciency (D ews e al., 2000). Fu he mo e, he inco - po a ion o a hyb idisa ion p obe in an ELISA o ma gene ally inc eases he speci ici y and sensi i i y o he PCR assay and elimina es analysis by gel elec opho - esis. Fig. 2. Analysis o con ol plasmid and esul an PCR p oduc s by aga ose gel elec opho esis. Lanes 1/2: DpnI-diges ed con ol (lane 1) and gB plasmids (lane 2), espec i ely; lane 3: P1/P2 PCR p oduc om con ol plasmid (460 bp); lane 4: XbaI diges ion o con ol P1/P2 amplicon (189 and 271 bp); lane 5: Ps I diges ion o con ol P1 /P2 amplicon (185 and 275 bp); lane 6: P1/P2 PCR p oduc om gB plasmid; lane 7: nega i e con ol; Lane 8: 10 kb molecula weigh ma ke . P. Daly, S. Doyle / Jou nal o Vi ological Me hods 107 (2003) 237/244 241 Based on PCR/ELISA esul s ob ained, he mini- mum de ec ion le el was es ima ed a 100 copies o gB plasmid and 63 genome equi alen s o EHV-1, espec- i ely. The sligh ly highe sensi i i y o de ec ion ob- se ed wi h EHV-1 DNA may e lec he p esence o non-speci ic ca ie DNA in he p epa a ion which p e en s abso p i e DNA loss on o ube walls. Compa - able ampli ica ion e iciencies o gB a ge and con ol, a iden ical copy numbe s, we e e iden which is a undamen al equi emen o he compe i i e PCR assay. In o de o de e mine he op imal copy numbe o con ol o be used in he compe i i e PCR/ELISA, compe i ion assays we e pe o med be ween he gB and con ol plasmids, espec i ely. Based on esul s ob- ained, 1000 copies o he con ol we e ound o be op imal o de ec ion o EHV-1 gB amplicons wi hou supp essing he sensi i i y o a ge de ec ion. Ba be e al. (1999) eco ded simila esul s using compe i i e in e nal con ol a 1000 copies in a PCR/ELISA o de ec human cy omegalo i us blood samples o hea and lung ansplan ecipien s. The clinical u ili y o he compe i i e PCR/ELISA assay was assessed by he co-ampli ica ion o 1000 copies o con ol wi h EHV-1 posi i e/nega i e e al Fig. 3. Compe i i e PCR/ELISA wi h (A) 10 000 o (B) 1000 copies o con ol plasmid in he p esence o a ying amoun s o EHV-1 gB plasmid. EHV-1 gB plasmid copy numbe anged om 10 2 /10 4 and was ampli ied alone (*/o*/) o in he p esence o con ol plasmid (da k and ligh boxes) a he a o emen ioned amoun s. The con ol DNP oligonucleo ide p obe was un eac i e o gB-de i ed amplicons (*/x*/). P. Daly, S. Doyle / Jou nal o Vi ological Me hods 107 (2003) 237/244242 issue samples. Posi i e EHV-1 e al issue samples (S726-8, S685-7 and 835-7) esul ed in he de ec ion o high le els o EHV-1 amplicons by PCR/ELISA using gB-speci ic DNP oligonucleo ide p obes while negligible le els o con ol amplicons we e eco ded. These esul s demons a e he compe i i e na u e o he empla es o he P1/P2 p ime s whe e he con ol was ou -compe ed by he high concen a ions o EHV-1 in posi i e issue samples. All h ee EHV-1 nega i e e al issue samples (S611-3, S723-5 and 597-9) analysed by PCR/ELISA Fig. 4. Compe i i e PCR/ELISA analysis o w EHV-1 a a ious dilu ions. Log dilu ions o pu i ied EHV-1 DNA we e ampli ied alone ( */o*/)o co-ampli ied wi h 1000 copies o he con ol plasmid (da k and ligh boxes). I is appa en ha high le els o EHV-1 DNA inhibi con ol ampli ica ion (Nea */10 3 dilu ions). Con ol ampli ica ion is e iden a dilu ions g ea e han 10 4 w EHV-1 DNA. The PCR/ELISA cu -o alue (0.140) was calcula ed om he mean nega i e specimen abso bance /2 s anda d de ia ions (0.095/2(0.0225); N/12). Fig. 5. E alua ion o EHV-1 posi i e and nega i e clinical specimens by PCR/ELISA. Con i med EHV-1 posi i e (S726-8, S685-7 and 835-7) and nega i e (S611-3, S723-5 and 597-9) issue samples, by immuno luo escence and i us isola ion, ga e he expec ed esul ollowing blinded es ing by PCR/ELISA. The e ec o he PCR inhibi o , NaI, was in es iga ed a 5 and 50 mM, espec i ely, in PCR eac ions con aining 1000 copies each o gB and con ol plasmid. No ampli ica ion was e iden a 50 mM NaI. The PCR/ELISA cu -o alue (0.140) is indica ed by he solid line. P. Daly, S. Doyle / Jou nal o Vi ological Me hods 107 (2003) 237/244 243 we e con ol amplicon posi i e and nega i e o gB amplicons. Thus, he nega i e issue samples we e ue nega i es due o he success ul ampli ica ion o he con ol. Howe e , nega i e samples S611-3 and S723-5 in he compe i i e PCR/ELISA had signi ican ly lowe abso bance eadings han ha o sample 597-9 and 1000 copies o con ol alone. This sugges s ha he e may be low concen a ions o PCR inhibi o s ex ac ed om hese issue samples. To demons a e he po en ial o he con ol in he PCR/ELISA o de ec po en Taq polyme ase inhibi o s, he e ec o NaI a di e en concen a ions was in es iga ed. The p esence o high concen a ion o NaI (50 mM) comple ely inhibi ed he ampli ica ion o gB and con ol as de e mined by he absence o signals on he ELISA. The e ec o educing he concen a ion o inhibi o (5 mM) elie ed he inhibi o y e ec NaI and esul ed in con ol de ec ion in he PCR/ELISA. Since EHV-1 is belie ed o pe sis ollowing ini ial in ec ion, he compe i i e PCR/ELISA de eloped he e could be used o de e mine he ac ual i al load in he p egnan ma e. Vi al loads may co espond o he s age o disease and could be used as a ma ke o disease p og ession. I is cu en ly unclea i se ial su eillance can be used o moni o he p og ess o EHV-1 in ec ions in ho ses and he EHV-1 PCR/ELISA p esen ed he e may p o ide a use ul ool o in es iga e his si ua ion. In his s udy, he de elopmen o he i s , in e nally- con olled, compe i i e PCR/ELISA assay o he diagnosis o EHV-1 in ec ion is desc ibed. The assay allows o mul iple sample de ec ion using end-s age, colou ime ic de ec ion o amplicons using gB ype- speci ic DNP oligonucleo ide p obe hyb idisa ion. The inclusion o a con ol elimina es he possibili y o alse nega i e EHV-1 esul s due o he co-ex ac ion o PCR inhibi o s. The design o his compe i i e PCR/ELISA diagnos ic assay allows o he apid and sensi i e de ec ion o EHV-1 in ho ses which, ollowing u he alida ion, may ha e signi ican po en ial o elimina - ing he occu ence o abo ion s o ms in s ud a ms. Acknowledgemen s We a e g a e ul o En e p ise I eland o unding his wo k (P ojec code: ST/99/115). We also hank D J. Sla e and D W. Eichho n o supplying specimens o analysis. Re e ences Allen, G.P., Yea gan, M.R., Tu inen, L.W., B yans, J.T., McCollum, W.H., 1983. 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