The p esen documen , in i s elec onic o m, is made a ailable o use s as an analy ical me hod. This
documen is he p ope y o ANSES. Any ep oduc ion, whe he o al o pa ial, is pe mi ed only on he
exp ess condi ion ha he sou ce is ci ed, o example by indica ing i s e e ence (including i s e sion
and yea ) and i s i le.
Funded by he Eu opean Union. Views and opinions exp essed a e howe e hose o he au ho (s) only
and do no necessa ily e lec hose o he Eu opean Union o Eu opean Heal h and Digi al Execu i e
Agency (HaDEA). Nei he he Eu opean Union no HaDEA can be held esponsible o hem
ANSES/PR3/7/01-04 [ e sion d]
ANSES/FGE/0139
ANALYTICAL METHOD FOR FOOD SAFETY
REFERENCE: ANSES/LSAlimen s/LSA-INS-1517- Ve sion 04
23 Oc obe 2023
GenoLis e ia Mul iplex: Iden i ica ion by mul iplex eal- ime PCR o 30
majo clonal complexes o Lis e ia monocy ogenes s ains
ANSES Labo a o y o Food Sa e y
Eu opean Union Re e ence Labo a o y o Lis e ia monocy ogenes
Ré é ence : LSA-INS-1517 - e ision 4
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His o y o he me hod
A me hod can be upda ed in o de o ake accoun o changes.
A change is conside ed majo when i ela es o key aspec s, he applica ion ield o he c i ical poin s o he
me hod, which i aken in o accoun could signi ican ly change he scope, he pe o mance o he esul o he
analy ical me hod. A majo change leads o majo adap a ions. I he me hod is modi ied o his ex en , i is
hen e alida ed, o ally o pa ially.
A change is conside ed mino i i p o ides use ul o p ac ical cla i ica ions, e o mula es he ins uc ions o
make hem clea e o mo e p ecise, o co ec s mino e o s. A mino change do no a ec he pe o mance
cha ac e is ics o he me hods; i does no equi e e alida ion
The ollowing able summa ises he e sion his o y o his me hod.
Ve sion
Na u e o he
changes
(majo /mino )
Da e
Main changes
V0
Ini iale e sion
17/01/2022
-
V1
Mino change
15/02/2023
Add epo ing dye and quenche in able 1 and sec ion 6
In e p e a ion p ecisions we e p o ided in sec ion 9.1
Add he possibili y o 15 µL eac ion in sec ion 8.1 and 8.2
Range o DNA concen a ion was upda ed o 1-0.1 ng/µL. I was speci ied ha
measu emen mus be ob ained by luo ime ic quan i ica ion o double s and
DNA
V2
Majo change
05/10/2023
Supp ession o CC14-ST91-ST360 om he me hod
Modi ica ion o he scheme o he me hod acco dingly, addi ion o CC18 wi h he
FAM dye o he duplex CC8-CC121 which become a iplex CC8-CC18-CC121
The numbe ing o he PCR was changed s a ing om IIa-5 because PCR IIa-4
(CC14-ST91-CC18) was emo ed in able 1
Mino change
The dye o CC18 p obe was changed o FAM (due o he modi ica ion o he
iplex IIa-1) and CC121 o HEX (due o ypo e o ) in able 1
Addi ion o a speci ic posi i e con ol o CC101 in sec ion 5.1 & 8.2
The sen ence: All con ols DNA can be p o ided by he EURL o Lm, was
added o sec ion 5.1
The sen ence “I he C is less han 30, he PCR is sys ema ically edone”. Was
emo ed om sec ion 9.1. As i comes in con adic ion wi h he cycle h eshold
(C ≤30) es ablish o he me hod.
Maisons-Al o labo a o y o ood sa e y was eplaced by ANSES labo a o y o
ood sa e y, exac da e o publica ion, men ion o EU unding and “F ench was
speci ied o he na ional e e ence labo a o y was added in on page.
V3
Mino change
23/10/2023
Re e ence o F ench NRL was emo ed om he on page.
V4
Mino change
19/09/2025
Add Table 5a wi h ecommended mas e mixes and add wo eal- ime PCR
mas e mixes wi h issues in able 5b, based on in e -labo a o y alida ion ail
esul s
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Sec ion 5.1: Modi ica ion o he posi i e con ol. Plasmid “pBluesc ip IISK+” was
eplaced by “pUC57”, and speci ic DNA con ol o CC101 was emo ed.
Me hod DNeasy® Blood & Tissue ki QiaGen was added in sec ion 7.3.
Majo change
Real- ime PCR 149_CC2_8862 pe o ms poo ly on complex DNA ex ac s (such
as en ichmen b o h ex ac s). I has been eplaced by a eal- ime PCR loca ed
a a nea by gene ic posi ion wi h simila cha ac e is ics, called 149_CC2_8868.
Modi ica ion o Tables 2, 3, and 6.
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Fo ewo d
This me hod has been de eloped by: Benjamin FELIX, Ka ine CAPITAINE and Sand ine TE
ANSES –Labo a o y o Food Sa e y (Labo a oi e de Sécu i é des Alimen s)
Eu opean Union Labo a o y o Lis e ia monocy ogenes
Ad esse : 14, ue Pie e e Ma ie Cu ie – 94700 Maisons-Al o
Con ac : Benjamin FELIX, eu l-li[email p o ec ed]
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Con en s
Fo ewo d ....................................................................................................................................... 4
Wa nings and sa e y p ecau ions ................................................................................................ 7
BIOHAZARD .................................................................................................................................. 7
HAZARDS OF REAGENTS ............................................................................................................ 7
1. Pu pose and scope ................................................................................................................ 8
2. Re e ence documen s ........................................................................................................... 8
3. Te ms, ac onyms and de ini ions ......................................................................................... 8
4. P inciple o he me hod ......................................................................................................... 9
5. Reagen s ................................................................................................................................ 9
5.1 Posi i e con ol ................................................................................................................... 9
5.2 PCR eagen s .................................................................................................................. 11
6. Equipmen and ma e ials .................................................................................................... 15
Usual mic obiology appa a us and equipmen ........................................................................ 15
7. Samples ................................................................................................................................ 15
7.1 Condi ions o accep ance o samples .............................................................................. 15
7.2 Conse a ion o samples be o e analysis ......................................................................... 15
7.3 Conse a ion o samples a e analysis ............................................................................ 16
7.4 P epa a ion o samples o analysis .................................................................................. 16
8. P ocedu e ............................................................................................................................. 16
8.1 Real ime PCR analysis in he o m o iplex/duplex PCRs .............................................. 16
8.2 P epa a ion o he PCR mix .............................................................................................. 17
8.3 Ampli ica ion condi ions .................................................................................................... 17
9. Resul s .................................................................................................................................. 18
9.1 Con ol o he alidi y o he esul s ................................................................................... 18
9.2 Calcula ion and exp ession o esul s ............................................................................... 18
10. Pe o mance cha ac e is ics o he me hod ....................................................................... 26
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Tables and igu e
Table 1: e e ence ADN ..................................................................................................................................................... 10
Table 2: P ime s and p obes o he de ec ion o GenoLis e ia a ge ed genes ............................................................. 122
Figu e 1: GenoLis e ia mul iplex Scheme ........................................................................................................................ 194
Table 3: Exp ession o he esul s...................................................................................................................................... 19
Table 4: Molecula se o ype in e p e a ion ...................................................................................................................... 20
Table 5: STs o CCs o he s ains ha can be de ec ed by he PCR o GenoLis e ia Scheme .......................................... 220
Table 6a & b: Recommended Mas e Mix & Mas e mix wi h PCR issues ........................................................................ 22
Table 7: Posi i e con ol plasmids composi ion ................................................................................................................ 23
In oduc ion
The “GenoLis e ia mul iplex” in house me hod was de eloped by he Salmonella and Lis e ia Uni
(SEL), his e sion is an adap a ion o he ANSES in e nal me hod LSA-INS-1493 de eloped o
simplex PCR assays hough high h oughpu PCR.
The p esen in house me hod “GenoLis e ia mul iplex” is made o 12 mul iplex eac ions.
The p esen in house me hod was de eloped by he ANSES Food Sa e y Labo a o y (ANSES LSAl)
in 2021-2022 as pa o a mul i-pa ne collabo a i e e o . The SEL uni , wi hin he amewo k o he
ac i i ies o he Eu opean Union Re e ence Labo a o y o Lis e ia monocy ogenes (EURL Lm), is
he leade o he p ojec . This me hod was de eloped in collabo a ion wi h he B3PA uni o he
ANSES LSAl Boulogne su Me loca ion, he Na ional Re e ence Labo a o ies (NRLs Lm) o he
Eu opean Union (BE, CH, CY, DE, GR, IT, NL (RIVM and WFSR), PT, SE, SI, and SK) in cha ge o
L. monocy ogenes yping in hei coun y, he F ench Ins i u e o Pig and Po k Indus y (IFIP) and
ADRIA de elopmen o Quimpe .
The me hod was published in 2023 in he scien i ic jou nal Mic obiology Spec um:
h ps://zenodo.o g/ eco ds/10017525.
The “GenoLis e ia mul iplex” includes he molecula se o yping scheme o Vi ullo e al. 2013. I
iden i ies 6 gene ic ma ke s h ough 2 iplex PCR eac ions (Lmo0737, p s, plcA and Lmo1118,
ORF2110, ORF2819) o p o ide he molecula se o ype o he s ains.
Following molecula se o yping, he me hod allows he iden i ica ion, by eal- ime PCR, o 30 clonal
complexes (CCs) o Mul i locus sequence yping (MLST) ci cula ing in ood, animals, and
en i onmen and esponsible o clinical cases in humans in Eu ope. The 30 CCs a e co e ed by 30
gene ic ma ke s pa o PCR eac ions p o iding he ollowing iden i ica ions: CC1, CC2, CC3, CC4,
CC5, CC6, CC7, CC8, CC11-ST451, CC14-ST14-ST206-ST399, CC18, CC19-ST398-802-1308,
CC20, CC21, CC26, CC29, CC31, CC37, CC54, CC59, CC77, CC87, CC101, CC121, CC155,
CC193, CC199, CC204, and CC224. CC9 iden i ica ion is p o ided by Lmo1118 molecula se o ype
PCR.
The p ime s and p obes we e de eloped bo h, o be pe o med as 35 simplex PCR (In house me hod
LSA-INS-1493) o one duplex and 11 iplex PCR in he p esen me hod.
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Wa nings and sa e y p ecau ions
The use o his me hod should be amilia wi h s anda d labo a o y p ac ices. I is he
esponsibili y o he use o es ablish p ope heal h and sa e y p ac ices and o ensu e
compliance wi h applicable egula ions.
I is essen ial ha manipula ions conduc ed in acco dance wi h his me hod be pe o med by
app op ia ely ained pe sonnel.
BIOHAZARD
In ec ion by his bac e ium is equen ly accompanied by mino diges i e p oblems, including
dia hoea. Howe e , he e a e se ious o ms ha may a ec sensi i e popula ions such as p egnan
women, he elde ly and he immunocomp omised indi iduals. Ope a o s handling L. monocy ogenes
mus he e o e wo k in a Le el 2 labo a o y.
HAZARDS OF REAGENTS
Fo all ques ions, consul sa e y da a shee s (SDS) p o ided by he p o ide o p oduce .
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1. Pu pose and scope
This me hod desc ibes he iden i ica ion, on L. monocy ogenes bac e ial s ains, o 30 CCs o MLST
and 5 molecula se o ypes by eal- ime PCR h ough 12 mul iplex PCR eac ions.
2. Re e ence documen s
[1] Vi ullo, M. e al. Real- ime PCRs assay o se og ouping Lis e ia monocy ogenes and di e en ia ion
om o he Lis e ia spp. Mol Cell P obes 27, 68-70 (2013).
[2] NF EN ISO 20837 s anda d: Mic obiology o ood and animal eeding s u s - Polyme ase chain eac ion
(PCR) o he de ec ion o ood-bo ne pa hogens - Requi emen s o sample p epa a ion o quali a i e
de ec ion
[3] NF EN ISO 22119 s anda d: Mic obiology o ood and animal eeding s u s - Real- ime polyme ase
chain eac ion (PCR) o he de ec ion o ood-bo ne pa hogens - Gene al equi emen s and de ini ions
[4] NF EN ISO 20838 s anda d: Mic obiology o ood and animal eeding s u s - Polyme ase chain eac ion
(PCR) o he de ec ion o ood-bo ne pa hogens - Requi emen s o ampli ica ion and de ec ion o
quali a i e me hods
3. Te ms, ac onyms and de ini ions
C Cycle h eshold
dATP Desoxyadenosine iphospha e
dCTP Desoxycy idine iphospha e
dGTP Desoxyguanosine iphospha e
dTTP Desoxy hymidine iphospha e
DNA Desoxy ibonucleic Acid
dNTP Desoxynucleo ide
Lm Lis e ia monocy ogenes
pb base pai
p s gene Gene coding o phospho ibosylpy ophospha e syn he ase
PCR Polyme ase Chain Reac ion
plcA Phospha idylinosi ol-phospholipase C
Taq The mus aqua icus
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4. P inciple o he me hod
The 35 GenoLis e ia genomic ma ke s scheme (Table 2) a e iden i ied by Taqman® eal- ime PCR
p obes. Two PCR p o ocols we e op imized, one con en ional he mal cycling and one as he mal
cycling (Needing dedica ed mas e mix and he mocycle ). The eal- ime PCR assay iden i y a
gene ic ma ke speci ic o he 30 CCs and 5 molecula se o ypes sough by he me hod. Among he
35 eal- ime PCR p obes o he GenoLis e ia scheme, 29 we e designed wi hin he amewo k o he
me hod de elopmen by he ANSES SEL uni . The eal- ime PCR assay also in eg a e he 6 ma ke s
o he molecula se o yping scheme o Vi ullo e al. 2013 ha iden i y he s ain molecula se o ype
(so called se og oup) IIa, IIb, IIc, IVb e IVa and L h ough wo iplex PCR eac ions. The PCR ha
iden i y he genus Lis e ia (p s – gene o he phospho ibosylpy ophospha e syn he ase) and a
species monocy ogenes (plcA – gene o he Phospha idylinosi ol-phospholipase C) o Vi ullo e al.
2013 scheme a e used as in e nal con ol. Bo h PCR a e pa o he same iplex. The Vi ullo e al.
2013 scheme was de eloped by he UK-NRL Lm, Public Heal h England. This me hod de ails he
implemen a ion o 12 mul iplex PCRs (adap ed o use o con en ional eal ime PCR ins umen s).
The molecula se o ype should be pe o med in a i s s age, he CC mul iplex iden i ica ion should
be pe o med acco ding o he molecula se o ype. In his me hod (Figu e 1), he CC a e gi en by
o de o abundance acco ding o h ee s udies ha desc ibed he di e si y o Lm in Food in F ance
(Mau y e al. 2016, Félix e al. 2018) and in Eu ope (Painse e al. 2019) (Figu e 1).
This me hod equi es he ollowing s eps:
▪ Ve i ica ion o he concen a ion and quali y o he DNA ex ac s
▪ Real- ime PCR analysis
▪ In e p e a ion o esul s
5. Reagen s
Cau ion: Ce ain ade names o supplie s may be men ioned by name in he desc ip ion o he
p oduc s necessa y o applying his me hod. This in o ma ion is gi en as an indica ion o use s o
he me hod and does no in any way imply ha ANSES ecommends he exclusi e use o hese
p oduc s. Equi alen p oduc s may be used i i has been demons a ed ha hey p oduce he same
esul s.
5.1 Posi i e con ol
The e e ence DNAs used as PCR posi i e con ols o each o he ma ke s come om s ains in he
ANSES collec ion, excep o wo s ains ha come om he Hunga ian NRL and Slo ak NRL
collec ions (Table 1). The 39 ampli ica ion p oduc s de i ed om he genome o he e e ence s ains,
lanked a each end by a 20 bp egion, we e cloned in o six puc57 plasmids. The plasmids ca y
be ween i e and nine ampli ica ion p oduc s and a e o ganized as ollows (Table 7):
- Plasmid DNA IIa A: CC8-CC121-CC18, CC31-CC37-CC155, CC7-CC14-ST14-CC204
- Plasmid DNA IIa B: CC20-CC21-CC101, CC26-CC29-CC193, CC199-CC11-ST451-CC19-
ST398
- Plasmid DNA IIb: CC3-CC87-CC5, CC59-CC77-CC224
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7.3 Conse a ion o samples a e analysis
The genomic DNA samples can be s o ed a e analysis in a DNA bank o la e use, no ably o he
sequencing o he comple e genome o he s ains ( alid i he ex ac ion was ca ied ou ollowing
he Wiza d® Genomic DNA Pu i ica ion Ki P omega ex ac ion (ANSES LSA-INS-1227 p ocedu e)
o DNeasy® Blood & Tissue ki QiaGen (ANSES LSA-INS-1494 ope a ing mode).
7.4 P epa a ion o samples o analysis
The genomic DNA ex ac s a e aliquo ed and dilu ed wi h DNase and RNase ee molecula
biology wa e o b ing hem o a DNA concen a ion be ween 1 and 0.1 ng/µL, be o e being added
o he PCR mix. DNA concen a ion should be ob ained by luo ome ic measu emen o he only
quan i ica ion o double s and DNA.
8. P ocedu e
8.1 Real ime PCR analysis in he o m o iplex/duplex PCRs
This me hod equi es o use con en ional eal ime PCR ins umen s wi h h ee de ec ion
channels: dye FAM 520 nm, dye HEX 554 nm (compa ible wi h VIC 549 nm), dye Cy5 669 nm. In
his analysis con igu a ion he PCRs a e pe o med as iplex FAM-HEX-Cy5 o duplex FAM-HEX.
The iplex mixes all ha e he same composi ion, as well as he duplex mixes. The eac ion olume
could be pe o med in 15 µL and 20 µL o ma .
The PCR should be pe o med in wo ounds:
1) Fo all samples iplex PCR 1 (p s, plcA, Lmo0737) and PCR 2 (ORF2819, ORF2110,
Lmo1118).
2) The o he iplex o duplex PCR (Table 2) may be pe o med acco ding o he molecula
se o ype deduced (Figu e 1). Mul iplex PCR can be pe o med one by one o se e al PCRs
can be pe o med a he same ime.
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8.2 P epa a ion o he PCR mix
In he mix PCR oom
Sample p epa a ion should be adap ed acco ding o he PCR mix used.
In a DNA oom
Acco ding o he pla e plan, add 2µl o each dilu ed DNA i he inal olume o eac ion is 20µL,
o 1.5µl i he inal olume is 15µL.
Con ols: Fo each duplex o iplex PCR, include:
- he posi i e con ol a 1X concen a ion (mix u e o plasmids) (Sec ion 5.1)
- he nega i e PCR con ol: DNA- ee wa e (sec ion 5.2).
8.3 Ampli ica ion condi ions
Con en ional p og am Fas p og am*
2 min
50°C
2min
95°C
10 min
95°C
3 sec
95°C
40 cycles
15 sec
95°C
40 cycles
30 sec
60°C
1 min
60°C
Tempe a u e amping 2°C/sec Tempe a u e amping 3 o 5 °C/sec
*Fas P og am was es ed wi h only GoTaq®
P obe qPCR Mas e Mix ( e P omega
A6102)
T iplex
Duplex
20 µL
15 µL
20 µL
15 µL
Reagen
µl / eac ion
µl / eac ion
Reagen
µl / eac ion
µl / eac ion
H2O
5.3
3.975
H2O
6.2
4.65
2X mas e mix
10
7.5
2X mas e mix
10
7.5
20 µM Fo wa d P ime 1
0.3
0.225
20 µM Fo wa d P ime 1
0.3
0.225
20 µM Re e se P ime 1
0.3
0.225
20 µM Re e se P ime 1
0.3
0.225
20 µM P obe 1
0.3
0.225
20 µM P obe 1
0.3
0.225
20 µM Fo wa d P ime 2
0.3
0.225
20 µM Fo wa d P ime 2
0.3
0.225
20 µM Re e se P ime 2
0.3
0.225
20 µM Re e se P ime 2
0.3
0.225
20 µM P obe 2
0.3
0.225
20 µM P obe 2
0.3
0.225
20 µM Fo wa d P ime 3
0.3
0.225
To al
18
13.5
20 µM Re e se P ime 3
0.3
0.225
20 µM P obe 3
0.3
0.225
To al
18
13.5
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9. Resul s
9.1 Con ol o he alidi y o he esul s
An ampli ica ion, o be conside ed posi i e, mus ha e a de ec ion h eshold less han o equal o 30
cycles (C ≤ 30). La e ampli ica ions, beyond 30 cycles (C >30), a e conside ed as non-speci ic.
The posi i e con ol and he nega i e con ol mus ha e he expec ed esul o alida e he
manipula ion (5.1 and 8.1.1). The in e nal PCR con ol p s is posi i e o he L. monocy ogenes,
L. innocua, L. welshime i, L. seelige i, L. i ano ii, L. ma hii and L. g ayi s ains (Vi ullo e al. 2013).
The PCR in e nal con ol plcA is posi i e o all L. monocy ogenes s ains.
The ampli ica ion cu e o he es samples, o be conside ed posi i e, mus be wi hin a de ec ion
in ensi y ange g ea e o equi alen o he posi i e con ol (ΔRN): PCR con ols (5.1) and in e nal
con ols p s and plcA (8.1.1). This con ol should be applied pa icula ly o he eal ime PCR
21322_SNP_LMO00485_CC101.
In e nal con ols (p s and plcA), mus be posi i e o all L. monocy ogenes. To be alid, he es PCRs
mus ha e a C equi alen o he in e nal con ols (p s and plcA). I he es PCR C is highe han
he in e nal con ols, i is necessa ily ela ed o con amina ion.
9.2 Calcula ion and exp ession o esul s
The in e p e a ion o he sample esul s is ca ied ou by compa ison wi h he con ol DNA acco ding
o able 4. The GenoLis e ia PCR has been de eloped so ha a s ain can only be iden i ied by one
o he PCRs pe o med. I a s ain is iden i ied by se e al PCRs, con amina ion by se e al di e en
DNA ex ac s mus be suspec ed.
I is possible ha a s ain wi h a di e en ST o CC om he a ge CCs may be iden i ied by some
o he PCRs in he GenoLis e ia scheme. These join iden i ica ions could no be a oided when
designing he p obes. They a e conside ed as c oss eac ions. A lis o he c oss eac ions iden i ied
du ing he de elopmen o he me hod is de ailed in able 5. The c oss eac ions mus be speci ied
when w i ing he analysis epo .
The in e p e a ion o he esul s is done acco ding o he able 3 and 4 o he exp ession o esul s.
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Table 3: Exp ession o he esul s
Clonal Complex GenoLis e ia
CC1
CC2
CC3
CC4
CC5
CC6
CC7
CC8
CC9
CC11 (ST451)
CC14 (ST14-ST206-ST399)
CC18
CC19 (ST398)
CC20
CC21
CC26
CC29
CC31
CC37
CC54
CC59
CC77
CC87
CC101
CC121
CC155
CC193
CC199
CC204
CC224
O he CCs*
Lis e ia no Lis e ia monocy ogenes**
« Non- Lis e ia »***
CC1-P-F-R (149_CC1_1911)
CC2-P-F-R (149_CC2_8868)
CC3-P-F-R (4711_CC3_6907)
CC4-P-F-R (4711_CC4_3572)
CC5-P-F-R (4711_CC5_9574)
CC6-P-F-R (149_CC6_4418)
CC7-P-F-R (258_CC7_6968)
CC8-P-F-R (258_CC8_952)
CC9-P-F-R (258_CC9_5506)
CC11-ST451_1_P-F-R (111_ CC451_9204)
CC14-ST14-P-F-R (258_SNP_LMO002932_ST14)
CC18-P-F-R (258_CC18_7946)
CC19-ST398-P-F-R (111_Lmo01705_ST398_1)
CC20-P-F-R (21322_CC20_1143)
CC21-P-F-R (21322_CC21_5690)
CC26-P-F-R (3511_CC26_4338)
CC29-P-F-R (3511_CC29_2081)
CC31-P-F-R (1615_CC31_8696)
CC37-P-F-R (1615_CC37_4509)
CC54-P-F-R (1_CC54_731)
CC59-P-F-R (359_ CC59_5912)
CC77-P-F-R (359_CC77_3146)
CC87-P-F-R (258_CC87_8655)
CC101-P-F-R (21322_SNP_LMO00485_CC101)
CC121-P-F-R (258_CC121_3159)
CC155-P-F-R (1615_CC155_5334)
CC193-P-F-R (3511_CC193_5118)
CC199_2-P-F-R (111_CC199_711)
CC204-P-F-R (258_CC204_2959)
CC224-P-F-R (359_CC224_7283)
plcA
p s
Posi i e PCR eac ion C ≤30
*: Includes all L. monocy ogenes no a ge ed by GenoLis e ia .
**: includes he ollowing specie L. g ayi, L. ma hii, L. innocua, L. welshime i, L. seelige i, L. innocua, L. seelige i, L. i ano ii
***: includes bac e ial s ains no belonging o he amily Lis e iaceae excep o he ollowing secies: L. boo ia, L. newyo kensis, L. ipa ia, L. g andensis, L.
weihens ephanensis, L. weihens ephanensis, L. ocou iae, L. aqua ica, L. lo idensis , L. Fleischmannii subsp. Fleischmanii, L. leischmanii subsp. Colo adonensis.
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Table 4: Molecula se o ype in e p e a ion
Molecula se o ype (Doumi h e al. 2010
nomencla u e)
Lmo0737
Lmo1118
ORF2110
ORF2819
plca
p s
IIa
IIb
IIc
IVb
IVb (a ypical)
IVa
L
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Table 5: STs o CCs o he s ains ha can be de ec ed by he PCR o GenoLis e ia
scheme
CC o ST a ge ed by
he me hod
Real ime PCR p obe
e e ence
Join de ec ion o s ains no belonging o he
a ge ed CCs – (co-de ec ion p edic ed in silico)
CC1
149_CC1_1911
ST213, CC183, ST773 (CC373*, ST1125)
CC2
149_CC2_8868
CC3
4711_CC3_6907
CC1000 (CC489, ST558, ST1046, ST1041, CC1211)
CC4
4711_CC4_3572
(ST631)
CC5
4711_CC5_9574
CC6
149_CC6_4418
CC7
258_CC7_6968
(CC373*)
CC8
258_CC8_952
ST1110 (ST1018)
CC9
Lmo1118
(ST184, ST395, ST1331*)
CC11-ST451
111_ CC451_9204
CC14-ST14-ST206-ST399
258_SNP_LMO002932_ST14
CC689 (ST843)
CC18
258_CC18_7946
CC19-ST398-802-1308
111_Lmo01705_ST398_1
(CC1127)
CC20
21322_CC20_1143
ST19, ST173 (ST226*, ST364, ST378, ST1021, ST1071, ST1078)
CC21
21322_CC21_5690
CC403
CC26
3511_CC26_4338
(ST376, ST790, CC912, ST1024, ST1331)
CC29
3511_CC29_2081
(CC344, ST1082)
CC31
1615_CC31_8696
CC37
1615_CC37_4509
CC321 (ST648, ST828, ST1068)
CC54
1_CC54_731
CC59
359_ CC59_5912
CC77
359_CC77_3146
CC87
258_CC87_8655
CC88
CC101
21322_SNP_LMO00485_CC101
(CC90, ST671, ST1127)
CC121
258_CC121_3159
CC689
CC155
1615_CC155_5334
CC193
3511_CC193_5118
CC124 (ST798)
CC199
111_CC199_711
(CC739, ST1331*)
CC204
258_CC204_2959
(ST798)
CC224
359_CC224_7283
ST581, ST585 (ST226*, ST1118)
*: ST o CC iden i ied as a c oss- eac ion by wo dis inc p ime and p obe se s
(): ST o CC unde b acke s we e p edic ed in silico as c oss eac ion, bu no es ed on s ain
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© Anses – 14, ue Pie e e Ma ie Cu ie - 94701 Maisons-Al o Cedex
Table 6a: Recommended PCR Mas e Mix
Mas e Mix
Manu ac u e
The mal cycling
Pe eCTa qPCR ToughMix Low Rox
Quan abio
Con en ional
SsoAd anced Uni e sal P obes Supe mix
Bio-Rad
GoTaq P obe qPCR mas e mix
P omega
Fas
Table 6b: Mas e Mix wi h PCR issues
Mas e Mix
Manu ac u e
The mal cycling
Taqman Uni e sal PCR mas e mix
Li e echnologies
Con en ional
Ligh cycle 480 P obes mas e
Roche
Pla inum Quan i a i e PCR Supe Mix UDG
In i ogen-The moFishe
Luna® Uni e sal P obe qPCR Mas e Mix
New England Biolabs
Uni e sal TaqMan Mul iplex Mas e Mix
The mo ishe
Fas
Kapa p obe as qPCR Ki Mas e Mix (2X) Uni e sal
Kapa Biosys ems
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Table 7: Posi i e con ol plasmids composi ion
Real ime PCR name
ANSES s ain
e e ence
Molecula
se o ype
MLST
Clonal
complex
MLST ype
Amplicon
size
Recombinan plasmid
e e ence
Sequence used as posi i e con ol (amplicons lanked by 20 bp a each end)
p s
17SEL375LM
IIc
CC9
ST622
141
Molecula _se o yping
aaa c aa cg gaac agc gaagaga gcgaaagaag agg a gag agggaaa caag g ac ca ag ga ggagaaa c
caaa aaca gaagaaag a ccg gg g ca g a a g a caa caacgag aa cc g aaaccagaa aa ggaa
plcA
17SEL375LM
IIc
CC9
ST622
79
Molecula _se o yping
cgc ggaagc ga aagcag c ggacaa c c gaa g cacac cggacca g ag ca c gaa ac gg agg gcgc
cgaac gca gccgaa gcg
Lmo0737
17SEL375LM
IIc
CC9
ST622
102
Molecula _se o yping
a ca gaaacaacaaaacgagcacggaag gc agg aacga g c aa cgc ggaaaa gggaaga gg a ga gagaaag
g gggcga ccac gc aaacaaga gcaaaagaag ga aagcaa
ORF2110
17SEL82LM
IVb
CC6
ST6
94
Molecula _se o yping
a agg ggaggaa g gcacaagcagcagaggaagccccaa cga gaaaaga ag ggggaaacgg aacaaa gacggagaa
gag a ag cga gaga ag ga agaaga g aa ca
ORF2819
10CEB88LM
IIb
CC3
ST3
134
Molecula _se o yping
aag caaacaa ccag aaca cac aaagcc ccca gagc c cg aaga cga a acg ca ggcag ccaggac cac g ca
c c c c ccg a a g ggagggag ggag a gcg ggaaa c ccgg gaaaag ag a a
149_CC1_1911
17SEL1LM
IVb
CC1
ST1
100
DNA-IVb
ga g gcaaa cagggg cga ag g ca aggagaaaa aa aac aaa gcga gca gag gc ggaaa aagg a gaaga
aaa accaa a gaa agagagc agaagcg aaa ga gag
149_CC2_8868
17SEL376LM
IVb
CC2
ST2
109
DNA-IVb
a aaaaa acaagccaga g ccc a caa c c c caaa caa c aa gg ga agaa ag cga aac agga g ggca
ag c g aaac agaa caag gca ca aaa c gc aaca c
4711_CC3_6907
10CEB88LM
IIb
CC3
ST3
119
DNA-IIb
ca c gcaaaacccaaa aga caaagca ac a aa ag cgc gacgaa a caaac cacaaa gc aac ca a
ca agg a a a ac caagaa agagagaa ccg ccca gc a gcc gag
4711_CC4_3572
17SEL543LM
IVb
CC4
ST4
129
DNA-IVb
a c c g a cc a aca cg agcc ca ca a gcc cc accaac g ac gaagga ggcagggagggccacc a ac aaa c
aa a agaa ac caa c ca ga a c aaa aa cc cgg ag ccg a ac acaa aaca
4711_CC5_9574
16SEL667LM
IIb
CC5
ST5
99
DNA-IIb
aag c aa aaa gagaa cc gc agc c g aga a aaa ac aaagacaca aa ccgc ggcaaagc gga ggcgcg gc
ag g c g aaaag acc cagaaac aa a c cgg
149_CC6_4418
17SEL82LM
IVb
CC6
ST6
127
DNA-IVb
aggaa gcacca a accccggcag g ga aca gggaaacgga c a aaacacgcaagcaaa agcaa a ca g ag aa ca
a c a aaacca c ccac g g c aaag aa c a c accagca cg gaaa ac
258_CC7_6968
16SEL17LM
IIa
CC7
ST691
138
DNA-IIa-A
c ca aa aac g c agaacc a a gaggca a aac gcaac ccagag caaca aa c gac g gaag ggg ccgaa gg
gccag ga a gc acc cacc agagaag agaaa cca ac ca a cacc caa g c aaaa
258_CC8_952
17SEL12LM
IIa
CC8
ST8
117
DNA-IIa-A
a gag a cc agga a gg acggg ag g aaag cacagaaac c aagccggaag g c ca g aa gaca aa a c g
a cc aaaaag caggaac cac ca gaaaaggc ca g ca aaa ca c
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Lmo1118, 258_CC9_5506
17SEL375LM
IIc
CC9
ST622
170
Molecula _se o yping
ag aaaga c g cc ag a ccagga aagacccc acc a c c cc gag g a acgcc ca aa gaaaaaagca cc c
ca c g a aagca caa gagagaga a gaaggag g cac ca ga cga caa gg c gg aaaaaac aa
a c g
111_ CC451_9204
20SEL24LMLM
IIa
CC11
ST451
167
DNA-IIa-B
c a g ca aaaga aaga gggag aa ga a gga aa ag aagac cgcgca g gc g gcacaaag ca c g aaa
aa gaaa ga ggagaa a ac a acagaa gggacaa aaaaaa ga ga ga c c g a aa caagaaagaa agg g aag
cac c gc ggaa
258_SNP_LMO002932_ST14
17SEL22LM
IIa
CC14
ST91
132
DNA-IIa-A
gaaaa a gag aaaacaacga gcaac gc a aggaaaaa g a caggacaaa cag gca ggcca cacggcaggc c acacc
aagcgaa g acgaac a ag ggaagaaa gaaa
258_CC18_7946
17SEL4LM
IIa
CC18
ST18
148
DNA-IIa-A
g gc aga g aa caag caca gg a a caag cagacaaaaac caca cga g c caac acagaag cgagg caga
a ga c a aaggaaaaaa gc c acacagaggag aga aaacaaac ag gaaa aaacc g a cag acaaa ccagaa
111-Lmo01705_CC19-ST398_1
21SEL232LM
(164 LNR HU)
IIa
CC19
ST398
134
DNA-IIa-B
c gga ca agaa g c gc ccgc ga a caa gac cacg gcaaa gaac cggcg ag a caa acg cgcaac agc
acggccgaaga gaa ggca gacaaa gcgcaag c gaccg g gggacgaa aa gaaacaaaa gg
21322_CC20_1143
08CEB286LM
IIa
CC20
ST20
121
DNA-IIa-B
cg c cg g cc aa ag g aagcaaa c agcc g caa c ga gga a aaca aaagcaggaacg caca
g aaaaa a c aaaa c ag g ca cacgaaa gca c ccaagaaaaa
21322_CC21_5690
17SEL88LM
IIa
CC21
ST21
111
DNA-IIa-B
aa c gc c ca cacc gcaac aaaa aac a c caac c a a aaaaa caac gc g aaaccaagaa aa caaa c aa
c caccaaaaac ca cag aa ca cca aa ac cc c a
3511_CC26_4338
09CEB411LM
IIa
CC26
ST26
131
DNA-IIa-B
gcagc a aaagaaaacacgacg a gac aaa cagaca aa gaa ca ggacgc c aaaaa g c aaaagaa aaagaa
gccaaacaaac aaa a ca aacaacaaac acga ac c g gaaga gc gg gaacaac aaac aca
3511_CC29_2081
17SEL50LM
IIa
CC29
ST849
114
DNA-IIa-B
caaaaacggaac caaaaacggc a aaacggaga a gacgc gaac gc aa gcac a accaaaaaagg a cg ac cga
c ga c aa aa a caccaac g ag aac gccaga ga a a ca ccc
1610_CC31_8696
17SEL370LM
IIa
CC31
ST31
148
DNA-IIa-A
c ggg aacagaa ga ccggag g a ggca a gaaag ca c a ca a gaaa cc aaa acaaaa g aagccaaac a
aaaaagcaa gaggaga agc cg ca a aaaga aa c c a caacga cca cca caa c ca ac c
1610_CC37_4509
17SEL105LM
IIa
CC37
ST37
114
DNA-IIa-A
ga g agcagaaaaagaagccagagaa ggc aga ac caggcagcac ca aa ccaga caga gcaggaggaaaagcaga a
aa gg gg a ggg ga aaagg a caa c c a gg cccaa ggaaa a agaa g
1_CC54_731
19SEL881LM
IVb
CC54
ST54
102
DNA-IVb
gggaacagcg aag agaggaca a aga g cg c ggacg gac a aaaca c gga cagg ga a accgg acgg acg
ggaggc a a gc aag g gg gaagca a c a gaagagga g
359_ CC59_5912
17SEL517LM
IIb
CC59
ST59
123
DNA-IIb
g a g aaagaaac ga cagcaaaagacagcaga aaa aaaaagaa c ccgacgaaacgc gcaa cc a aga acaagaa
gaaaaa gc g g cagaaaaag aag aaa a a c ggc a c ga cgcc a ga
359_CC77_3146
09CEB593LM
IIb
CC77
ST77
141
DNA-IIb
ga g gga ggaaaaaccacgaa caaac g gaac a cagga c g gacagaaccaa cc ccaaccaaagaaggg acaaa c
a gg gg a ga gcaaaaacaaacggaac aag gggaa caaca ga aaaa gcc gcgaagaa a cac a acgcgca
258_CC87_8655
17SEL107LM
IIb
CC87
ST87
124
DNA-IIb
c gaaccgaaa agaggg gacacca g aaa c cga cgcaaaa cc gag ga aaaca cgcc ac cgaaaag gaa gaaa
cccgcca gc ccaacaca a aaa aa ca ccaag c gca agc aaccaa cagc
21322_SNP_LMO00485_CC101
03EB425LM
IIa
CC101
ST775
100
DNA-IIa-B
aa g gg c ccaaaagaaa ggcac gaa a caaacca g a gaaagaac ag ggacgcggc agcaca aaca aagag
gc aaacg aaaa cgaacg a ggc ccagaaa c gggacg a
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258_CC121_3159
17SEL63LM
IIa
CC121
ST121
146
DNA-IIa-A
cagcaaac ggccaa a a ggc ac gaa a a cccca a a caaggc a ga ggaaaaaa g acaca gga a a acagc
c aaaa ggcgg ag agcaa c aaaaaaaa ag aca acagc agaaca a aa ga aaa ccgag aa gg aa c
1610_CC155_5334
17SEL412LM
IIa
CC155
ST155
133
DNA-IIa-A
aga agg g accaaaag g cagag cgaa ca aaaaaggcaaaagcaaaa caca caa c aaaa g ga a c cag cac
gaaaa agcgcaaa aggga gga c gaa a caa ac gaaaa ccaga cagaaaa a gaaca ga
3511_CC193_5118
11CEB245LM
IIa
CC193
ST193
133
DNA-IIa-B
agaaaaacccc g ca g g a cc gacagggg ga gaggaacca a ca ccaa gaaa caaaca acag agaaaaaca
ga a cgg a cccca ca g a a gac cg a cccac caaccg agc gg ggc ac cg aa
111_CC199_711
20SEL79LM
IIa
CC199
ST199
193
DNA-IIa-B
aa gag a aa gaa cggagca cac a a ca acaagca ga aa c agac c ccac ccagcaaacgc c g a aa ac
gacccga cc ca aaaa g c ag gg cggacaa a cgcgcg agaa a g ga ac a gaa ca ca a gg c a
aca ccaac gaccaca gac ca caga
258_CC204_2959
17SEL510LM
IIa
CC204
ST204
101
DNA-IIa-A
ac caaaa c ca ga cc c gg ac c aaa a ca c a caa c caaa g ggacaac c c aa ca c aa aaaa aaca
ggga aa c cggc c ga cc aac caaca a
359_CC224_7283
10CEB615LM
IIb
CC224
ST224
106
DNA-IIb
caaagaaaag caaa agagaagga a agaaa gaaag ag aaa a aaaga ac acga aa ag gaaacaa ggacaa
ga agg a cagc ac agagaga acg caaaaa caa c aaaaa
