Ci a ion: Regal, P.; Do al, A.;
Ga cía-Ramos, I.; Cepeda, A.;
Ga ido-Maes u, A.; Lamas, A.
Loop-Media ed Iso he mal
Ampli ica ion-Based Wo k low o he
De ec ion and Se o yping o
Salmonella spp. in En i onmen al
Poul y Flock Samples. Foods 2024,13,
4069. h ps://doi.o g/10.3390/
oods13244069
Academic Edi o : Ewen C. D. Todd
Recei ed: 18 No embe 2024
Re ised: 4 Decembe 2024
Accep ed: 13 Decembe 2024
Published: 17 Decembe 2024
Copy igh : © 2024 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
A icle
Loop-Media ed Iso he mal Ampli ica ion-Based Wo k low o
he De ec ion and Se o yping o Salmonella spp. in
En i onmen al Poul y Flock Samples
Pa icia Regal 1, Anne Do al 1, I ia Ga cía-Ramos 1, Albe o Cepeda 1, Alejand o Ga ido-Maes u 2,*
and Alexand e Lamas 1,*
1Food Hygiene, Inspec ion and Con ol Labo a o y (LHICA-USC), Depa men o Analy ical Chemis y,
Nu i ion and B oma ology, Facul y o Ve e ina y Science, Campus Te a, Uni e sidade de San iago de
Compos ela (USC), 27002 Lugo, Spain; [email p o ec ed] (P.R.); [email p o ec ed] (A.D.);
[email p o ec ed] (I.G.-R.); [email p o ec ed] (A.C.)
2Labo a o y o Mic obiology and Technology o Ma ine P oduc s (Mic oTEC), Ins i u o de In es igaciones
Ma inas (IIM—CSIC), Edua do Cabello, 6, 36208 Vigo, Spain
*Co espondence: [email p o ec ed] (A.G.-M.); [email p o ec ed] (A.L.)
Abs ac : Salmonella spp. is one o he mos impo an oodbo ne pa hogens wo ldwide. Gi en he
ac ha poul y and poul y p oduc s a e he main sou ce o human in ec ion, Salmonella con ol in
hese a ms is o u mos impo ance. To be e con ol his pa hogen in a ms, boo swabs a e used o
sample a m en i onmen s bu he analysis o hese swabs is mainly based on cul u e-dependen
me hods. In he p esen s udy, a no el loop-media ed iso he mal ampli ica ion (LAMP) me hod
was de eloped o he apid sc eening o Salmonella spp. in boo swab samples om b oile lock
en i onmen s. Fou di e en DNA ex ac ion p o ocols we e e alua ed in dep h, including a simple
he mal lysis, a chelex-based p o ocol and wo he mal lysis p o ocols ollowed by he pu i ica ion
o magne ic beads made o silica (“glass milk”) in o de o de e mine he mos sui able al e na i e
o po en ial on-si e, a m analyses. The me hodology e alua ion included a blind in e labo a o y
assay and as a p oo -o -concep , a naked-eye colo ime ic assay was also included. Following he
inal me hodology, i was possible o each an LoD
50
o 1.8 CFU/25 g o he samples, wi h a high
ela i e sensi i i y (95.7%), speci ici y (100%) and accu acy (96.6%) along wi h Cohen’s kappa o
conco dance wi h espec o he ISO s anda d 6579-1:2017 o 0.9, wi h an RLOD o 1.3. In addi ion o
his, due o he ele ance o ce ain se o ypes wi h he genus Salmonella spp., a se o ype LAMP panel
o he speci ic iden i ica ion o S. Typhimu ium, S. En e i idis, S. In an is, S. Hada and S. Vi chow
was also de eloped. E en hough some deg ee o c oss- eac i i y among he p ime s de eloped
was obse ed, all he se o ypes could be accu a ely iden i ied based on hei mel cu e analysis
p o ile. Taken oge he , in he p esen s udy, a apid Salmonella spp. sc eening me hod, sui able o
a m applica ions, was de eloped, along wi h a se o yping panel ha could be used in a labo a o y
se up o he iden i ica ion o he mos ele an se o ypes o he genus, aking ad an age o eal- ime
ampli ica ion ollowed by mel cu e analysis.
Keywo ds: loop-media ed iso he mal ampli ica ion; Salmonella spp.; S. Typhimu ium; S. En e i idis;
S. In an is; S. Hada ; S. Vi chow; poul y; en i onmen ; a m
1. In oduc ion
The poul y ma ke , h ough he p oduc ion o mea and eggs, is one o he mos
impo an sec o s wo ldwide due o i s in luence in global ma ke s [
1
]. Addi ionally, his
indus y plays a c ucial ole in main aining ood sa e y. Poul y p oduc s a e one o he
main sou ces o Salmonella spp. in he ood chain. The con ol o his pa hogen is ca ied ou
om p ima y p oduc ion o e ail ma ke s. Based on he Eu opean Union Regula ion (EC)
No 2160/2003 [
2
], membe s a es ha e de eloped hei na ional plans o Salmonella con ol
Foods 2024,13, 4069. h ps://doi.o g/10.3390/ oods13244069 h ps://www.mdpi.com/jou nal/ oods
Foods 2024,13, 4069 2 o 17
in poul y p oduc ion. These plans include en i onmen al sel -moni o ing sampling by
he ope a o o de e mine he p esence o Salmonella spp. in a ms. One sampling me hod,
exclusi ely used in he case o b oile s, in ol es walking inside he b eeding acili y wi h
boo swabs, which a e la e analyzed o he p esence o Salmonella spp. [
3
]. This p ocedu e
has been epo ed o be he mos sensi i e and cos -e ec i e app oach o he de ec ion o
Salmonella spp. in a ms [
4
,
5
]. In addi ion o he genus, ce ain se o ypes a e conside ed
pa icula ly ele an o human heal h, namely S. Typhimu ium, S. En e i idis, S. In an is,
S. Vi chow, and S. Hada [6].
The ISO 6579-1:2017 is he e e ence me hod o Salmonella de ec ion in he ood
chain [
7
]. This s anda d applies o ood and en i onmen al con ol in he ood indus y, as
well as en i onmen al samples om p ima y p oduc ion. The me hod is based on classical
mic obiology, in ol ing wo en ichmen s eps, selec i e media seeding, and con i ma ion
h ough biochemical me hods. A minimum o h ee days is needed o classi y a sample as
nega i e and a minimum o i e days o de e mine i a sample is posi i e. These u na ound
imes a e u he ex ended due o se o yping using an ise a. This delays he p oduc ion
sys em, and may pose a challenge o an indus y ope a ing on na ow economic ma gins.
Al e na i e me hods o he de ec ion o Salmonella spp., such as immunoassays like
he VIDAS o molecula me hods, ha e been de eloped in ecen decades [
8
–
10
]. Molecula
me hods, pa icula ly eal- ime PCR (qPCR), ha e gained impo ance in pa hogen de ec ion
in he ood chain as hey allowed us o o e come he limi a ions o classical, cul u e-based
me hods [
11
,
12
]. While hese me hods s ill equi e a p io en ichmen s ep, he imes a e
signi ican ly sho ened, and esul s can be ob ained wi hin 24 h. Al hough qPCR is he
gold s anda d among molecula ampli ica ion me hods, i has some limi a ions, p ima ily
equi ing ela i ely complex equipmen capable o empe a u e amping and luo escence
de ec ion [13,14].
In ecen yea s, a se ies o molecula iso he mal ampli ica ion echniques, such as
Recombinase Polyme ase Ampli ica ion (RPA) o loop-media ed iso he mal ampli ica ion
(LAMP), ha e eme ged [
15
,
16
]. In hese echniques, ampli ica ion occu s a a cons an
empe a u e in a simple he mal block o wa e ba h. Mo eo e , hese echniques suppo
he in oduc ion o chemicals ha enable isual de ec ion wi hou he need o luo escence
measu emen [17,18].
The cu en s udy p esen ed wo main goals. The i s was o de elop a LAMP-based
me hod o de ec he p esence o Salmonella in en i onmen al samples om poul y a ms
in less han 24 h, along wi h a p oo o concep o a low-cos , colo ime ic o ma o u u e
labo a o y assay decen aliza ion. The second goal consis ed o a subsequen s ep o be
pe o med on Salmonella-posi i e samples, and was ocused on a panel o independen
LAMP assays o apid iden i ica ion o he p esence o some o he mos ele an se o ypes
o he poul y indus y, namely S. Typhimu ium, S. En e i idis, S. In an is, S. Vi chow, and
S. Hada .
2. Ma e ials and Me hods
2.1. Bac e ial S ains and Cul u e P epa a ion
All bac e ial s ains used in he p esen wo k a e lis ed in Table 1. Speci ically,
Salmonella En e i idis WDCM 00030 was selec ed as he e e ence s ain o he de el-
opmen and op imiza ion o he LAMP assays o he de ec ion o Salmonella spp., as well
as he S. En e i idis se o ype-speci ic LAMP. S. Vi chow LHICA C11/22, S. In an is LHICA
C12/20, S. Hada LHICA C11/21, and S. Typhimu ium WDCM 00031 we e used o de-
elop he co esponding se o ype-speci ic LAMP assays. Addi ional Salmonella se o ypes,
as well as non-Salmonella bac e ial species, we e used o inclusi i y/exclusi i y assays.
All s ains we e conse ed in c yo ials a
−
20
◦
C. The p epa a ion o esh cul u es was
pe o med by ans e ing one c yoball o a 25 mL lask wi h 10 mL o B ain Hea In usion
(BHI, Me ck Millipo e, Bu ling on, MA, USA) and incuba ed wi h agi a ion (150 pm) a
37
◦
C du ing 18 h. Upon comple ion, he s ains we e pla ed in nu ien aga (NA, VWR,
Ba celona, Spain) and incuba ed a 37
◦
C o 24 h. Finally, he pla es we e conse ed and
Foods 2024,13, 4069 3 o 17
4
◦
C un il use. Fo spiking assays, Salmonella s ains we e g own as desc ibed and he
cul u e was se ially dilu ed and samples inocula ed wi h di e en olumes o di e en
dilu ions. Also, dilu ions we e pla ed in NA o de e mine bac e ial concen a ion. Bu e ed
Pep one Wa e (BPW, Me ck Millipo e, Da ms ad , Ge many) was used o sample en ich-
men . Semisolid Rappapo Vassiliadis medium (MRVS, Di coTM, BD, Mad id, Spain) was
used o Salmonella selec i e en ichmen . Xylose Lysine Deoxychola e aga (XLD, The mo
Scien i ic, Oxoid) and RAPID’Salmonella aga (Bio-Rad, He cules, CA, USA) we e used o
Salmonella isola ion. Media we e used acco ding o ISO 6579-1:2017 [19].
Table 1. Bac e ial s ains o inclusi i y and exclusi i y es s.
S ain S ain Ta ge
STM4497 sa A G oup_29846 G oup_21126 G oup_27174
S. Typhimu ium
WDCM 00031 + + - - - -
C13/22 + + - - - -
C3/21 + + - - - -
C4/20 + + - - - -
C4/22 + + - - - -
C7/21 + + - - - -
C9/20 + + - - - -
S. Typhimu ium monophasi
C15/22 + + - - - -
C9/21 + + - - - -
C9/22 + + - - - -
C7/20 + + - - - -
S. En e i idis
C1/20 + - + - - -
C1/21 + - + - - -
C10/20 + - + - - -
C10/21 + - + - - -
C2/22 + - + - - -
C7/22 + - + - - -
S. Hada
C11/21 + - - + - -
C2/20 + - - + - -
C3/22 + - - + - -
S. In an is C12/20 + - - - + -
C2/21 + - - - + -
S. Vi chow
C11/22 + - - - - +
C15/20 + - - - - +
C6/21 + - - - - +
S. F esno C13/20 + - - - - -
S. Lawndale C6/20 + - - - - -
S. Abony C11/20 + - - - - -
S. Agama C15/21 + - - - - -
S. Agbeni C13/21 + - - - - -
S. Augus enbo g C12/22 + - - - - -
S. Be a C14/21 + - - - - -
C8/22 + - - - - -
S. Coeln C8/20 + - - - - -
S. Dublin C1/22 + - - - - -
C5/21 + - - - - -
S. Gi e C5/20 + - - - - -
S. Glouces e C4/21 + - - - - -
S. Lagos C10/22 + - - - - -
S. S anley C12/21 + - - - - -
S. S anley C5/22 + - - - - -
S. Tedding on C3/20 + - - - - -
Foods 2024,13, 4069 4 o 17
Table 1. Con .
S ain S ain Ta ge
STM4497 sa A G oup_29846 G oup_21126 G oup_27174
S. We nige ode C14/22 + - - - - -
C8/21 + - - - - -
S. Yo uba C14/20 + - - - - -
B. ce eus WDCM 00151 - NT NT NT NT NT
E. aecalis
20825 - NT NT NT NT NT
CECT 481 - NT NT NT NT NT
WDCM 00009 - NT NT NT NT NT
A. baumannii CECT 452 - NT NT NT NT NT
Klebsiella pneumoniae CECT 8453 - NT NT NT NT NT
S. ube is CECT 994 - NT NT NT NT NT
S. agalac iae CECT 183 - NT NT NT NT NT
S. dysgalac iae CECT 758 - NT NT NT NT NT
L. monocy ogenes
WDCM 00110 - NT NT NT NT NT
WDCM 00021 - NT NT NT NT NT
L1AM0 - NT NT NT NT NT
L. innocua CUP 1375 - NT NT NT NT NT
P. ae uginosa WDCM 00024 - NT NT NT NT NT
P. luo escens WDCM 00115 - NT NT NT NT NT
P. agi WDCM 00116 - NT NT NT NT NT
E. coli
CECT 99 - NT NT NT NT NT
AMC 76 - NT NT NT NT NT
CECT 5947 - NT NT NT NT NT
C179-12 - NT NT NT NT NT
C. di icile CECT 531 - NT NT NT NT NT
S. au eus WDCM 00034 - NT NT NT NT NT
CECT 54 - NT NT NT NT NT
Y. en e ocoli ica WDCM 00038 - NT NT NT NT NT
WDCM 00039 - NT NT NT NT NT
C. jejuni AMC - NT NT NT NT NT
C. coli UM - NT NT NT NT NT
AMC - NT NT NT NT NT
CECT: Spanish Type Cul u e Collec ion. WDCM: Wo ld Da a Cen e o Mic oo ganisms. AMC: collec ion om
he Ins i u e o Applied Mic obiology, ASMECRUZ. UM: Uni e si y o Minho. CUP: Ca holic Uni e si y o Po o.
NT: No es ed.
2.2. P ime Design
Fo he de ec ion o S. Vi chow, S. Hada , S. In an is and Salmonella spp., new se s
o p ime s we e designed wi h P ime Explo e V5 (h ps://p ime explo e .jp/e/index.
h ml, 16 Decembe 2024). Fo he de ec ion o Salmonella spp., he p ime s epo ed by
Cos a-Ribei o e al., a ge ing he gene, we e selec ed [
20
], while o he se o ypes
Typhimu iums and En e i idis, he p ime s desc ibed by Azinhei o e al. we e selec ed and
a ge ed he genes STM4497 and sa A, espec i ely [
21
]. In Table 2, a comple e lis o all he
p ime s used in he p esen s udy is p o ided.
Fo he design o he new se s o p ime s, he e e ence genomes e ie ed om Re Seq
we e used, including NZ_CP025094.1 (g oup_27174), NZ_CP121068.1 (g oup_21126), and
NZ_CP022069.2 (g oup_29846) o S. Vi chow, S. Hada , and S. In an is, espec i ely.
Foods 2024,13, 4069 5 o 17
Table 2. Bac e ia, gene ic a ge s and p ime sequences.
Bac e ia Ta ge P ime Name Sequence (5′→3′) Re e ence
Salmonella spp.
FIP_ GCA TCA GCC AAC ATA GCG CCA
CTA CGC CAT CCG TTA TCA CA
[20]
BIP_ TCA GGT ACA AAC CGT CCC CAA G
CAT CCG TTC CGC CTG GTA
F3_ ACA CTG CTG TTC TGT AGC CT
B3_ AGG TGC CGA GAA TAG CCA
LF_ CCA GCA GGA CGC GTC TT
LB_ CGC GCA ATT TAA CCC TTA CTC G
S. Typhimu ium STM4497
FIP_STM ACC TGC AGC TCA TTC TGA GCA G
TCA AAA ACA ACG GCT CCG G
[21]
BIP_STM GAA AAG GAC CAC AAG TTC GCG C
TCA GTG AGC ATG TCG ACG AT
F3_STM AGC CGC ATT AGC GAA GAG
B3_STM GCG GTC AAA TAA CCC ACG T
S. En e i idis sa A
FIP_SEN AGC CCA CAG TGA GTA TCG TG CGC
TGC TGG TAG TGC ATG G
[21]
BIP_SEN CAG AGG TCA TGG CGC GCA AAT
GGC ATT GGT ATC AAA GGT GA
F3_SEN GTT GCT AAC ACG ACA CTG GAC
B3_SEN GTG GGA TAT TCT GAG CCC CTA T
S. In an is g oup_21126
FIP_INF
ATA GCC CAC CCC GCA ATT TCG GAC
TAC ATA CCG TAG CCC CA
Cu en
s udy
BIP_INF CCA GGCG AAT TAG TAT ACG ACC CAT
TTG AGC CAA GCT TCG AGG A
F3_INF GCA GAT ATC CCA TTA AAA ACT GAG C
B3_INF CGG TAC CAA TAG TAT CCC TAC CT
LF_INF GGG CGC ATC TTC CCA ATG
LB_INF TTT GTG GTT CTG GTA CTG TGC
S. Vi chow g oup_27174
FIP_VIR
TGG GCC AGC ACA AAT GAA TAC TGT G
CCA TGA TGG CAA CGG GAT
Cu en
s udy
BIP_VIR
TTA GGT GGC ACC CAT CCA GTG TAA
GGC AGC TCA CAA CGC
F3_VIR TGT ACC TGG TGT TTG ATA TTT CGT
B3_VIR CTG CAA TTG ACC AGT CGG T
LF_VIR TGG ATC TTA AAT AGT CAT CAA ACG A
LB_VIR CTG AAA CTT TTA TTT ATG CTT GGG T
S. Hada g oup_29846
FIP_HAD GCC GTG ATT TTC TTG ACT AAT TGA T
CAT GTG GCA ACA TTA GAA CG
Cu en
s udy
BIP_HAD TCT TTG GCG AGA AAA CAG CAA
TCC TTC ATA AAC GGA ACC G
F3_HAD AGA AGT CCG AGA GGA TGA
B3_HAD ACA GAT TAA GTT CCC TTC CAA
LF_HAD GCA TAC TGA AGC TCT TTT TCT GC
LB_HAD ATT TGC ATT GCT GGC GT
ep esen s a polyT linke be ween F2 and F1c, and B2 and B1c.
2.3. Nucleic Acid Ex ac ion
In he p esen wo k, ou di e en DNA ex ac ion me hods we e e alua ed om
simple he mal lysis o o he me hods ha include di e en ypes o pu i ica ion. In all
cases, 1 mL o en iched sample was ans e ed o a 1.5 mL mic o ube and cen i uged a
900
×
g o 1 min o elimina e sample deb is (s ep omi ed o pu e bac e ial cul u e). The
supe na an was ans e ed o a new mic o ube and cen i uged a 16,000
×
g o 1 min.
The supe na an was disca ded, and he pelle was used o DNA isola ion. All he DNA
samples we e conse ed a −20 ◦C un il use.
Foods 2024,13, 4069 6 o 17
2.3.1. The mal Lysis (TL)
The pelle was esuspended in 100
µ
L o nuclease- ee wa e and hea ed a 99
◦
C o
5 min a 1400 pm in a hea e block (The momixe , Eppendo AG, Wesseling-Be zdo ,
Ge many). Then, he sample was cen i uged again a 16,000
×
g o 1 min o elimina e
bac e ial and sample deb is and he supe na an was ans e ed o a new mic o ube.
2.3.2. Chelex
The pelle was esuspended in 100
µ
L o 6% Chelex
®
100 (Bio-Rad Labo a o ies, Inc.,
He cules, CA, USA). The sample was incuba ed o 15 min a 56
◦
C and 1400 pm, and hen
hea ed a 99
◦
C o 8 min a 1400 pm. Then, he samples we e cen i uged a 16,000
×
g o
1 min and he supe na an was ans e ed o a new ube.
2.3.3. The mal Lysis wi h Magne ic Bead Pu i ica ion
DNA was isola ed as desc ibed in Sec ion 2.3.1, bu , in his case, he inal supe na an
was mixed wi h 100
µ
L o magne ic beads (Mag-Bind
®
To alPu e NGS, Omega-Bio ek,
No c oss, GA, USA). The mix u e was incuba ed o 5 min a oom empe a u e. The
beads we e eco e ed wi h a magne ic pa icle concen a o (Dynal
®
MPC, In i ogen,
Ca lsbad, CA, USA) un il he liquid was clea . The supe na an was emo ed, and he
pelle was washed wo imes wi h 200
µ
L o 70% e hanol. Finally, he pelle was ai -d ied
o elimina e he es o he e hanol lea ing he caps open and he ubes in he magne ic
ack. The mic o ubes we e e ie ed om he magne ic ack and he magne ic pelle was
esuspended in 100
µ
L o nuclease- ee wa e . Sample was incuba ed o 2 min a oom
empe a u e and hen he ubes we e placed again in he magne ic ack. When he liquid
con aining he eleased DNA was clea , i was ans e ed o a new mic o ube.
2.3.4. The mal Lysis wi h “Glass Milk” Pu i ica ion (GM)
This DNA ex ac ion was based on he me hod desc ibed by Page, Robe , e al. wi h
some modi ica ions. In his case, he pelle was esuspended in 100
µ
L o nuclease- ee
wa e and 100
µ
L o a 4% SDS solu ion and incuba ed o 5 min a 99
◦
C and 1400 pm in a
hea e block. Then, 400
µ
L o 100% isop opanol, 200
µ
L o 1.25 M NaCl and 10
µ
L o “glass
milk” we e added and he sample was incuba ed o 5 min a oom empe a u e. A e ha ,
i was cen i uged o 15 s in minicen i uge a 2000
×
g. The supe na an was disca ded,
and he pelle was washed wo imes wi h e hanol a 70%. Then, he pelle was ai d ied
in a hea e block a 65
◦
C o 5 min wi h lead. A e ha , he pelle was esuspended in
100
µ
L nuclease- ee wa e o elease he DNA om he silica. The sample was cen i uged
a 2000×gand he supe na an was ans e ed o a new ube.
2.4. DNA Concen a ion and Quali y
The compa ison among he ou DNA ex ac ion p o ocols was pe o med by quan i-
ying he DNA concen a ion in all he samples spiked wi h Salmonella. The quan i ica ion
was pe o med wi h he dsDNA B oad Range (BR) assay ki (In i ogen™, The moFishe
Scien i ic, Wal ham, MA, USA) in combina ion wi h he comme cial luo ome e Qubi (In-
i ogen™, The moFishe Scien i ic, Wal ham, MA, USA) and DNA pu i y was de e mined
wi h NanoD op Li e Plus (The mo Scien i ic, Wal ham, MA, USA).
2.5. -LAMP
2.5.1. Real-Time -LAMP
Fluo escen LAMP assays we e pe o med in a Quan S udio 12k Flex Real Time PCR
sys em (Applied Biosys ems, The moFishe Scien i ic, Wal ham, MA, USA). P ime s we e
designed by a ge ing he gene. Reac ions we e ca ied using 12
µ
L Fas Mas e Mix
(ISO-004, Op iGene, UK), 800 nM o FIP/BIP p ime s, 400 nM o LB/LF p ime s and
200 nM o F3/B3 p ime s, 50 nM o CXR Re e ence Dye (P omega, Madison, WI, USA)
and 2
µ
L o empla e DNA and he emaining olume was comple ed wi h nuclease- ee
wa e . Technical duplica es we e pe o med o all samples, and he expe imen s we e un
Foods 2024,13, 4069 7 o 17
a 65
◦
C o 30 min, wi h luo escence acquisi ion e e y 30 s. Then, mel cu e analysis
was pe o med as ollows: samples we e hea ed a 95
◦
C o 1 s, 80
◦
C o 20 s, and hea ed
up o 95
◦
C wi h empe a u e inc emen s o 0.05
◦
C/s and luo escence acquisi ion a e
each empe a u e inc emen . Only samples wi h bo h posi i e echnical eplica es and Tm
alues alling wi hin he calcula ed a e age empe a u e
±
i s s anda d de ia ion we e
conside ed posi i e.
2.5.2. Colo ime ic -LAMP
Colo ime ic LAMP assays we e pe o med in 1.5 mL mic o ubes in a hea e block
(The momixe ) a 65
◦
C o 30 min. The eac ion composi ion and olume we e he same
as he luo escen LAMP bu wi hou ROX. A e illing 1.5 mL mic o ubes, he op o he
ube was co e ed wi h Pa a ilm
®
lea ing an opening o 1–2 mm on he hinge side o he
ube. Fi s , 1
µ
L o SYBR G een I 1000X (In i ogen
™
, The moFishe Scien i ic, Wal ham,
MA, USA) was deposi ed in he cen e o Pa a ilm
®
. Then, he lid was ca e ully closed,
and he ubes we e incuba ed. A e ha , ubes we e shaken o mix he sample wi h
SYBR G een I and cen i uged o 10 s in a minicen i uge (mySPIN 6, The moScien i ic,
Wal ham, MA, USA). Posi i e samples we e g eenish while nega i e samples emained
o ange. Fu he mo e, unde UV ligh , posi i e samples emi ed luo escence, while he
nega i e ones did no .
2.5.3. Se o yping LAMP
Once he p esence o Salmonella was con i med, i.e., -LAMP posi i e, he se o yping
LAMP was applied. A eal- ime, luo escence-based LAMP o de e mine he i e se o ypes
o impo ance in poul y p oduc ion (S. Typhimu ium, S. En e i idis, S. In an is, SVi chow,
S. Hada ) was de eloped. The lis o LAMP p ime s designed o his pu pose a e included
in Table 2. The eac ion condi ions we e he same as ha o he -LAMP. The accep ance
c i e ia we e he same de ined as hose o he -LAMP in Sec ion 2.5.1.
2.6. LAMP Valida ion
2.6.1. E alua ion o he Inclusi i y and Exclusi i y
Fo inclusi i y assays, 54 Salmonella s ains belonging o 31 di e en se o ypes and
3 di e en subspecies we e included. Fo exclusi i y assays, s ains om 28 o he bac e ial
s ains, belonging o 18 di e en species, we e included. In Table 1, a de ailed lis o he
mic oo ganisms included in he p esen s udy is p o ided. Pu e cul u es we e p epa ed as
desc ibed in Sec ion 2.1, and he DNA o his pu e cul u e was ex ac ed wi h he me hod
desc ibed in Sec ion 2.3.1.
2.6.2. Dynamic Range
The dynamic anges co e ed wi h he di e en p o ocols desc ibed in Sec ion 2.2
we e e alua ed wi h pu e DNA ex ac ed om he s ain WDCM 00030, as well as wi h a
eces-spiked sample. A e p epa ing he pu e cul u e, and he spiked sample, he DNA
was ex ac ed wi h all he DNA ex ac ion p o ocols, i was quan i ied, en- old se ially
dilu ed in nuclease- ee wa e (P omega, Madison, WI, USA) and analyzed in echnical
iplica es by -LAMP.
Rega ding he chicken eces, 100
µ
L o Salmonella o e nigh pu e cul u e was dilu ed
in 900
µ
L o eces in BPW. DNA was isola ed wi h he ou me hods desc ibed in Sec ion 2.2.
Then, isola ed DNA was se ially dilu ed as o he pu e cul u e.
2.6.3. De e mina ion o he Limi o De ec ion (LoD) and Rela i e Limi o
De ec ion (RLOD)
The Limi o De ec ion (LoD) wi h con idence o 50% (LoD
50
) and 95% (LoD
95
) was
de e mined o he ou DNA ex ac ion me hods es ed as desc ibed by Wil ich and
Wil ich [
22
]. Rega ding he Rela i e Limi o De ec ion (RLOD), he model desc ibed by
Mă gă i escu and Wil ich was used [
23
,
24
]. To de e mine hese limi s, chicken bedding
Foods 2024,13, 4069 8 o 17
was collec ed om a chicken a m and he absence o Salmonella was de e mined by ISO
6579-1:2017. Two pai s o boo swabs we e placed in a s omache bag wi h 25 g o chicken
bedding collec ed and spiked wi h di e en concen a ions o Salmonella En e i idis WDCM
00030. Then, samples we e homogenized wi h 225 mL o p e-wa med BPW and incuba ed
o 18 h a 37
◦
C. A e ha , 1 mL o he sample was collec ed and p ocessed as indica ed
in Sec ion 2.2 o DNA isola ion wi h he ou di e en me hods es ed. In addi ion,
he samples we e analyzed by he e e ence me hod ISO 6579-1:2017. B ie ly, a e BPW
incuba ion, 0.1 mL was ans e ed o modi ied MRVS. Pla es we e incuba ed a 41.5
◦
C o
48 h. Suspec ed samples we e s eaked in XLD and RAPID’ Salmonella pla es and incuba ed
o 24 h a 37
◦
C. Samples wi h p esump i e Salmonella colonies we e con i med wi h he
la ex agglu ina ion es (Mic ogen Biop oduc s L d., Su ey, UK).
2.6.4. Fi ness- o -Pu pose
Once he LoD
50
wi h each di e en DNA ex ac ion me hod was de e mined, all he
samples abo e he co esponding alue we e conside ed and classi ied as being in Posi i e
o Nega i e Ag eemen (PA/NA) i he -LAMP esul ma ched ha ob ained by he ISO
e e ence me hod and we e conside ed o be Posi i e o Nega i e De ia ions (PD/ND) i
he esul s did no ma ch he e e ence me hod. Once classi ied, hese alues we e used o
de e mine he ela i e sensi i i y, speci ici y and accu acy (SE, SP and AC, espec i ely)
along wi h Cohen’s kappa o conco dance (k) as p e iously desc ibed by Ande son e al.
and Tomás e al. [8,25].
The de eloped LAMP me hod was also es ed wi h in e labo a o y es s ca ied ou
annually by he Cen al Ve e ina y Labo a o y in Spain. This es includes en samples
o chicken eces inocula ed wi h 100 CFU, 10 CFU o no inocula ed. The samples we e
analyzed ollowing he p o ocol p e iously desc ibed and e alua ing he ou DNA isola-
ion me hods desc ibed. The inal wo k low was also used o analyze he ou ine samples
analyzed in he labo a o y as pa o he na ional Salmonella con ol plan.
2.7. G aphical Rep esen a ion and S a is ical Analysis
The s a is ical analyses and he ep esen a ion o he da a ob ained in he p esen
s udy we e pe o med wi h G aphpad P ism 10 (Bos on, MA, USA). One-way ANOVA
analysis wi h Dunn’s es was used o de e mine he exis ence o di e ences be ween
g oups (p< 0.05).
3. Resul s
3.1. LAMP Assay E alua ion
3.1.1. Inclusi i y/Exclusi i y
The e alua ion o he inclusi i y indica ed ha all he 31 se o ypes and 54 s ains
es ed in he p esen s udy epo ed posi i e esul s o he gene wi h an a e age mel ing
empe a u e (Tm) o 88.94
±
0.18
◦
C. Rega ding he e alua ion o he exclusi i y, a panel o
28 s ains co e ing 18 di e en species ypically encoun e ed in ood and en i onmen al
samples we e es ed. None o he exclusi i y panel s ains epo ed posi i e esul s, hus
demons a ing he speci ici y o he assay.
When ocusing on he se o ype-speci ic assays, i was possible o ampli y he di e en
se o ypes when using he co esponding se o ype-speci ic LAMP assay. In his sense, he
STM4497 (Typhimu ium) gene epo ed an a e age Tm alue o
86.98 ±0.28 ◦C
(10 s ains
including monophasic a ian s), sa A (En e i idis) epo ed alues o
86.19 ±0.37 ◦C
(6 s ains), and o g oup_29846 (Hada , 3 s ains), g oup_21126 (In an is, 2 s ains) and
g oup_27174 (Vi chow, 3 s ains), he Tm alues we e 83.46
±
0.35
◦
C, 86.64
±
0.21
◦
C
and 84.08
±
0.28
◦
C, espec i ely, as shown in Figu e 1. When ocusing on he exclusi i y,
20 non- a ge s ains we e es ed co e ing 16 di e en se o ypes, which we e all posi i e
o bu all nega i e o he se o ype-speci ic LAMP assays (no e ha all assays we e
in ended o be un in simplex o ma o a oid Tm misiden i ica ion).
Foods 2024,13, 4069 9 o 17
Foods 2024, 13, x FOR PEER REVIEW 9 o 18
en i onmen al samples we e es ed. None o he exclusi i y panel s ains epo ed posi-
i e esul s, hus demons a ing he speci ici y o he assay.
When ocusing on he se o ype-speci ic assays, i was possible o ampli y he diffe -
en se o ypes when using he co esponding se o ype-speci ic LAMP assay. In his sense,
he STM4497 (Typhimu ium) gene epo ed an a e age Tm alue o 86.98 ± 0.28 °C (10
s ains including monophasic a ian s), sa A (En e i idis) epo ed alues o 86.19 ± 0.37
°C (6 s ains), and o g oup_29846 (Hada , 3 s ains), g oup_21126 (In an is, 2 s ains) and
g oup_27174 (Vi chow, 3 s ains), he Tm alues we e 83.46 ± 0.35 °C, 86.64 ± 0.21 °C and
84.08 ± 0.28 °C, espec i ely, as shown in Figu e 1. When ocusing on he exclusi i y, 20
non- a ge s ains we e es ed co e ing 16 diffe en se o ypes, which we e all posi i e o
bu all nega i e o he se o ype-speci ic LAMP assays (no e ha all assays we e in-
ended o be un in simplex o ma o a oid Tm misiden i ica ion).
Figu e 1. G aphical summa y o he ypical Tm alues ob ained o he and se o yping LAMP
assays. Each assay was un in simplex o ma .
3.1.2. DNA Ex ac ion P o ocol Compa ison and -LAMP Dynamic Range
The dynamic ange o LAMP assay was de e mined in pu e DNA and eces, inocu-
la ed wi h S. En e i idis WDCM 00030. In bo h cases, he DNA was isola ed wi h he ou
me hods desc ibed. When analyzing he pu e bac e ial DNA, all ou p o ocols eached
he ange o he picog ams. In his sense, wi h TL and GM, he lowes concen a ion was
0.4 pg/µL, while wi h he beads, he alue sligh ly dec eased down o 0.2 pg/µL and wi h
chelex, a alue o 0.1 pg/µL was eached, as shown in Figu e 2A. In he case o chicken
eces inocula ed wi h Salmonella, diffe ences we e obse ed among he diffe en DNA ex-
ac ion me hods. I was de e mined ha he sample was spiked wi h 8.3 log CFU o Sal-
monella. Th ee p o ocols, namely chelex, TL and magne ic beads, we e able o ca y ou
de ec ion un il 3.3 log CFU/mL, while he GM me hod ca ied ou de ec ion un il 4.3 log
CFU/mL, as shown in Figu e 2B.
Figu e 1. G aphical summa y o he ypical Tm alues ob ained o he and se o yping LAMP
assays. Each assay was un in simplex o ma .
3.1.2. DNA Ex ac ion P o ocol Compa ison and -LAMP Dynamic Range
The dynamic ange o LAMP assay was de e mined in pu e DNA and eces, inocula ed
wi h S. En e i idis WDCM 00030. In bo h cases, he DNA was isola ed wi h he ou me hods
desc ibed. When analyzing he pu e bac e ial DNA, all ou p o ocols eached he ange o
he picog ams. In his sense, wi h TL and GM, he lowes concen a ion was 0.4 pg/
µ
L,
while wi h he beads, he alue sligh ly dec eased down o 0.2 pg/
µ
L and wi h chelex,
a alue o 0.1 pg/
µ
L was eached, as shown in Figu e 2A. In he case o chicken eces
inocula ed wi h Salmonella, di e ences we e obse ed among he di e en DNA ex ac ion
me hods. I was de e mined ha he sample was spiked wi h 8.3 log CFU o Salmonella.
Th ee p o ocols, namely chelex, TL and magne ic beads, we e able o ca y ou de ec ion
un il 3.3 log CFU/mL, while he GM me hod ca ied ou de ec ion un il 4.3 log CFU/mL,
as shown in Figu e 2B.
Foods 2024, 13, x FOR PEER REVIEW 10 o 18
Figu e 2. Dynamic ange co e ed wi h he diffe en DNA ex ac ion p o ocols wi h he -LAMP
assay wi h pu e DNA (A) and wi h bac e ia inocula ed in boo swabs (B). The ampli ica ion ime is
p o ided as T , Time o Th eshold.
3.2. DNA Ex ac ion P o ocol Compa ison
In o de o be e de e mine he pe o mance o each ex ac ion p o ocol, spiked sam-
ples we e used. The e we e no signi ican diffe ences in he quan i y o DNA isola ed be-
ween he diffe en ex ac ion p o ocols es ed (see Figu e 3A). Con a y o he DNA con-
cen a ion, when he pu i y o he ex ac s was measu ed, signi ican diffe ences we e ob-
se ed, i.e., he A260/A280 a io o magne ic beads (1.935 ± 0.084) and GM (1.899 ± 0.080)
was signi ican ly highe (p < 0.05) han he a io o chelex (1.685 ± 0.351) and TL (1.612 ±
0.441). The same esul s we e obse ed wi h he a io A260/A230, as shown in Figu e 3B,C.
(A)
(B)
Figu e 2. Dynamic ange co e ed wi h he di e en DNA ex ac ion p o ocols wi h he -LAMP
assay wi h pu e DNA (A) and wi h bac e ia inocula ed in boo swabs (B). The ampli ica ion ime is
p o ided as T , Time o Th eshold.
3.2. DNA Ex ac ion P o ocol Compa ison
In o de o be e de e mine he pe o mance o each ex ac ion p o ocol, spiked
samples we e used. The e we e no signi ican di e ences in he quan i y o DNA iso-
la ed be ween he di e en ex ac ion p o ocols es ed (see Figu e 3A). Con a y o he
Foods 2024,13, 4069 16 o 17
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