VORTRAGSTHEMA als 35pt Arial Black - EURL

VORTRAGSTHEMA als 35pt Arial Black - EURL

FEDERAL INSTITUTE
FOR RISK ASSESSMENT
Rapid detection and
specific differentiation of
Salmonella Enteritidis,
Salmonella Typhimurium and
its monophasic variant
by multiplex real-time PCR
Dr. Sven Maurischat
Outline
• Requirements according to Commission
Regulation (EC) No 2073/2005 and 1086/2011
• Development of a rapid detection / differentiation
assay
• Genetic variablitiy of monophasic S.
Typhimurium 1,4,[5],12:i:-
Sven Maurischat, EURL-Salmonella workshop XIX in Zaandam, the Netherlands
Page 2
Requirements regarding Salmonella in poultry
Sven Maurischat, EURL-Salmonella workshop XIX in Zaandam, the Netherlands
Page 3
Development of a multiplex real-time PCR Assay
for Salmonella serovar detection & differentiation
1. Selection of potential target genes –
Sybr®Green assays
1)
Sybr®Green Assay
R
R
R
2. Development of TaqMan singleplex assays
3. Development of a TaqMan multiplex assay
(adaptation of singleplex reactions)
2)
TaqMan® Assay
4. Validation of multiplex assay according to ISO 16140
R
3)
R
Sven Maurischat, EURL-Salmonella workshop XIX in Zaandam, the Netherlands
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Multiplex real-time PCR assay
5plex TaqMan assay
•
Specific detection of Salmonella Typhimurium and Salmonella Enteritidis
•
Differentiation of bi- and monophasic S. Typhimurium 1,4,[5],12:i:-
Target genes:
1.
S. Enteritidis: safA (SEN0281)
2.
S. Typhimurium: fliA-IS200
3.+ 4. 2nd H-Phase of S. Typhimurium (not expressed by monophasic variant):
3. fljB-hin region
4. hin-iroB intergenic region
5.
Internal amplification control (IAC): pUC18/19
Sven Maurischat, EURL-Salmonella workshop XIX in Zaandam, the Netherlands
Page 5
Target gene: S. Enteritidis - safA
• absent oder divergent in other Salmonella serovars
• chromosomally encoded on SPI-6
• coding a lipoprotein as fimbrial subunit
tnpA (pseudo)
safA safB
safC
safD
145 bp PCR Produkt
S. Enteritidis (9,12:g,m:-)
S. Blegdam (9,12:g,m,p:-)
S. Moscow (9,12:g,q:-)
Other serovars
Sven Maurischat, EURL-Salmonella workshop XIX in Zaandam, the Netherlands
Page 6
Target gene: S. Typhimurium (including monophasic STM)
• Intergenic region between IS200 und fliA Gen (Burnens et al. 1997)
Sigmafactor σ28
fliA
N-methylase-gene
fliB
IS200
254 bp PCR product
S. Typhimurium
bi- + monophasic variant
Other serovars
Sven Maurischat, EURL-Salmonella workshop XIX in Zaandam, the Netherlands
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Target genes: differentiation of bi- and monophasic STM
• Seroformula 1,4,[5],12:i:1,2
1,4,[5],12:i:-
• Different clonal lineages
• Different types of deletions possible
fljA
fljB (1,2)
hin
iroB
iroC
Aldridge P D et al. PNAS
2006;103:11340-11345
PCR 1 (307 bp) PCR 2 (264 bp)
H2 positive
H2 positive
Interrupted gene structure
Sven Maurischat, EURL-Salmonella workshop XIX in Zaandam, the Netherlands
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Monophasic S. Typhimurium 1,4,[5],12:i:• Spanish clone
16 Gene
17 Gene
55 Gene
Laorden L et al. J Clin Microbiol. 2010; 48(12): 4563–4566.
Sven Maurischat, EURL-Salmonella workshop XIX in Zaandam, the Netherlands
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Monophasic S. Typhimurium 1,4,[5],12:i:• US - clone
Soyer Y et al. J. Clin. Microbiol. 2009;47:3546-3556
Sven Maurischat, EURL-Salmonella workshop XIX in Zaandam, the Netherlands
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Monophasic S. Typhimurium 1,4,[5],12:i:• European clone
– 148 monophasic STM strains
– 70 % DT193
– 19 % DT120
- 57 % of DT120 strains (16 isolates)
were fljB PCR positive (2nd H-Phase)!
 min. 6 types of deletions
fljA
fljB
hin
1
Type 1
Type 2
Type 3
Type 4
iroB
2
iroC
ID
hin
06-04419
06-04525
06-04963
06-04991
07-00244
07-00528
07-00711
07-01243
07-01526
07-01577
07-01962
07-03017
07-03022
07-03136
07-04553
08-03970
08-03972
08-03977
08-03987
08-03989
08-04016
08-04030
+
+
Microarray
fljA
flj 1,x
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
H2PCR Eli
Type 5
Sven Maurischat, EURL-Salmonella workshop XIX in Zaandam, the Netherlands
Page 11
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Phagentyp
DT120
DT120
RDNC
DT120
DT59
DT120
DT120
nt
DT120
DT120
DT120
DT120
DT120
DT120
DT120
DT193
Combination of target genes: Multiplex real-time PCR Assay
• 5-plex Assay:
-
S. Typhimurium (IS200-fliB)
-
S. Typhimurium monophasic variant (2 targets)
-
S. Enteritidis (safA)
-
Internal amplification control (pUC19 specific sequence) (not shown)
S. Typhimurium bi- + monophasic
S. Enteritidis
S. Typhimurium biphasic
S. Typhimurium biphasic
Sven Maurischat, EURL-Salmonella workshop XIX in Zaandam, the Netherlands
Page 12
Limit of Detection
• Analytical detection limit: ~10 copies of DNA
10-100 cfu / ml
1
1
0,9
0,8
Detection Probability
Detection Probability
0,8
0,7
0,6
0,5
0,4
0,3
0,6
0,4
0,2
0,2
0,1
0
0
1
10
100
1000
10000
Kopien / Rxn
100000
1000000
1
10
100
1000
10000
Copies / reaction
predicted
obs (gesamt)
Cell Concentration
[cfu/ml]
safA (obs.)
fliA-IS200
(obs.)
S. Enteritidis (safA)
S. Typhimurium (fliA-IS200)
2nd H-Phase S. Typhimurium (fljB-hin)
2nd H-Phase S. Typhimurium (hin-iroB)
Sven Maurischat, EURL-Salmonella workshop XIX in Zaandam, the Netherlands
Page 13
100000
1000000
cfu / ml
H2 vorn (obs.)
H2 hinten (obs.)
PCR Efficiency
• Efficiency: 95-105%, R² > 0,995
S. Enteritidis
S. Typhimurium bi- + monophasic
S. Typhimurium 2nd H-phase
S. Typhimurium 2nd H-Phase
Sven Maurischat, EURL-Salmonella workshop XIX in Zaandam, the Netherlands
Page 14
Validation according to ISO 16140 : 2003 / 2008(E) for qualitative
methods
Rel. accuracy (naturally or artificially contaminated samples) RM / AM
•
20 samples / matrix (50% positiv, 50% negativ, if possible)
•
Calculation of rel. accuracy, sensitivity, specificity
Level of Detection (LOD) RM / AM (artificially cont. samples)
•
3 contamination level:
negative: 5 samples
1 - 10 cfu: 20 samples
10 - 100 cfu: 5 samples
Inclusivity / Exclusivity (only AM)
•
Inclusivity (min. 100 strains)
•
Exclusivity (min. 30 strains)
Ring trial (min. 10 valid data sets)
Sven Maurischat, EURL-Salmonella workshop XIX in Zaandam, the Netherlands
Page 15
Validation
• Inclusivity / Exclusivity
456 strains
serotyping
rtPCR
S . Typhimurium
2.H-Phase (1,2)
S . Enteritidis
+
-
+
-
+
-
S . Typhimurium (biphasic)
60
59
1
59
1
0
60
S . Typhimurium (monophasic)
111
111
0
2
109
0
111
S . Enteritidis
105
0
105
0
105
104
1
S . Typhiumurium-like serovars
42
1*
41
n.a.
n.a.
0
42
S . Enteritidis-like serovars
37
0
37
n.a.
n.a.
2**
35
other relevant S. serovars
61
0
61
n.a.
n.a.
0
61
other species
40
0
40
n.a.
n.a.
0
40
* S . Farsta
** S . Moscow / S . Blegdam
serovar
seroformula
no PCR pos strains / total no strains
serovar
no
PCR
pos
strains
/ total
strains
Typhimurium
1,4,[5],12:i:1,2
serovar
seroformular
no PCR
pos
strains
/ total
nono
strains
Salmonella
Typhimurium
PK
Agama
4,12:i:1,6
0/4
Enteritidis
9,12:g,m:Salmonella
Enteritidis
PK
1,4,[5],12:y:1,2
0/4
Coeln
Berta
9,12:g:0/3
Campylobacter
coli
0/1
Essen
4,12:g,m:0/1
Blegdam
9,12:g,m,q:1/1
Campylobacter
jejuni
0/1
Farsta
4,12:i:e,n,x
Dublin
9,12:g,p:0/81/2
Campylobacter
lari
0/1
1,4,12,27:i:l,w
Gloucester
0/1
Eastbourne
9,12:e,h:1,5
0/4
Citrobacter
diversus
0/1
1,4,[5],12:z10:1,2
Haifa
Gallinarum
9,12:-:0/40/4
Citrobacter
freundii
0/2
1,4,[5],12:g,m,s:[1,2]
Hato
0/1
Pullorum
9,12:-:0/4
Escherichia
coli
O22:H8
0/1
1,4,[5],12:r:1,2
Heidelberg
Gateshead
9,46:g,s,t:0/10/5
Escherichia
coli9,12:k:O26:H11
0/1
1,4,[5],12,27:l,[z13,
Goverdhan
0/1
0/1
Kunduchi
Escherichia
coli9,12:e,h:O91:H11
0/1
[z28]:1,2
Israel
0/1
Escherichia
coli9,12:l,z28:1,5
O113:H21
0/1
1,4,[5],12:i:1,5
Lagos
0/2
Javiana
0/2
Escherichia
coli
O128
0/1
1,4,[5],12:b:1,2
Paratyphi
B
Moscow
9,12:g,q:1/10/4
Escherichia
coli
O157
0/1
1,4,[5],12:e,h:1,2
Saintpaul
0/4
Naestved
9,12:g,p,s:0/1
Escherichia
fergusonii
0/1
1,4,[5],12,27:d:1,2
Stanley
Napoli
9,12:l,z13:0/10/4
Enterobacter
aerogenes
0/3
1,4,12:i:e,n,z15
Tsevie
0/2
Panama
9,12:l,v:1,5
0/2
Enterobacter
agglomerans
0/2
1,4,12:i:z6
Tumodi
Rostock
9,12:g,p,u:0/20/1
Enterobacter
amnigenus
0/1
Veneziana
11:i:e,n,x
0/1
Wangata
9,12:z4,z23:0/1
Enterobacter cloacae
0/3
Enterobacter gergoviae
0/1
Enterococcus faecalis
0/2
Klebsiella oxytocan
0/1
Klebsiella pneumoniae
0/2
Proteus mirabilis
0/2
Proteus penneri
0/1
Proteus vulgaris
0/1
Providencia alcalifaciens
0/1
Pseudomonas aeruginosa
0/1
Serratia marcescens
0/1
Shigella flexneri 2a
0/1
Yersinia enterocolitica
0/1
Yersinia intermedia
0/1
Yersinia pseudotuberculosis
0/1
Yersinia rohdei
0/1
Sven Maurischat, EURL-Salmonella workshop XIX in Zaandam, the Netherlands
Page 16
Validation
• Sensitivity / Specificity on broiler neck skin
Artificially spiked neck skin
o/n culture in 1% BPW at 37°C
serovar
[cfu/25 g]
S . Typhimurium
Cold stress 24 h at 4°C
S . Enteritidis
Dilution to 10-50 cfu / ml
no pos samples / total samples
level of contamination
cultural detection with
rtPCR
RVS
MKTTn
10-100
5/5
5/5
5/5
1-10
20/20
20/20
18/20
0
0/5
0/5
0/5
10-100
5/5
5/5
5/5
1-10
20/20
20/20
20/20
0
0/5
0/5
0/5
 100% rel. accuracy
 100% rel. sensitivity
 100% rel. specificity
Spiking of neck skins with 100 µl
(= 1-5 cfu / 100 µl), 5min incubation
1/10 dilution in 1% BPW
Homogenisation for 1 min
DNA-extraction with Chelex100
rtPCR
Pre-enrichment 18 ± 2 h at 37°C
Cultural detection (ISO 6579) + serotyping
Sven Maurischat, EURL-Salmonella workshop XIX in Zaandam, the Netherlands
Page 17
Summary
• Development of a highly specific multiplex rtPCR assay for the detection
of S. Typhimurium / 4,[5],12:i:- and S. Enteritidis
• More reliable differentiation on mono- / biphasic S. Typhimurium than
any up til now published PCR method
• Validated for analysis of pure bacterial cultures as well as the control of
chicken carcass contamination / poultry food control
• Suitable alternative to ISO 6579 based cultural detection method but…
•
cheaper
•
faster
•
fully comprehensive (e.g. atypical S. Typhimurium strains)
 Ring trials
Sven Maurischat, EURL-Salmonella workshop XIX in Zaandam, the Netherlands
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Aphasic S. Typhimurium
• „O-form“ or aphasic strains:
•
1,4,[5],12:-:-
•
4,[5]:-:-
•
4,12:-:-
• Serotyped as Salmonella Serogroup B or Salmonella Subspecies I
→ not restricted by EU regulations
serovar
STM-pos. PCR samples / total samples
4,5:-:-
3/5
4,12:-:-
9/15
4,5,12:-:-
8/9
69% S. Typhimurium
Sven Maurischat, EURL-Salmonella workshop XIX in Zaandam, the Netherlands
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Acknowledgments
 Dr. Burkhard Malorny
 Beatrice Baumann
FEDERAL INSTITUTE
FOR RISK ASSESSMENT
 Serotyping group
Dr. Sven Maurischat
Federal Institute for Risk Assessment
Max-Dohrn-Str. 8-10  10589 Berlin, GERMANY
Tel. 0 30 - 184 12 - 14 50  Fax 0 30 - 184 12 - 29 53
[email protected]  www.bfr.bund.de