Evaluation of the Salmonella / O157 & STEC screening GeneDisc Pack and the
EHEC Identification GeneDisc Pack for the detection of Shiga toxin-producing
Escherichia coli in samples of animal feces and raw meats
AE Heuvelink, W Lubberts, JTM Zwartkruis, R Dijkman, CAM van Heerwaarden, E de Boer
Food and Consumer Product Safety Authority (VWA), The Netherlands
Introduction
Materials & methods
The GeneDisc Cycler is an automated, miniaturized real-time PCR system.
It performs gene amplification in a disposable device, the GeneDisc, which
is preloaded with the reagents necessary for the reaction.
Samples: animal feces & raw meats
Enrichment (18-20 h): mTSB+n (20 mg/l) at 41.5°C & BPW at 37°C
PCR screening:
DNA extraction: Chelex-based
Cyclers: GeneDisc cycler & Lightcycler 2.0; same primers/probes
Target genes:
stx1 & stx2, eae, rfbE (O157), Salmonella
O26 (wxz), O103 (galE), O111 (wbd1), O145 (ihp1), H7 (fliC H7)
Cultural method:
Aim
Evaluation of:
(1) the Salmonella / O157 & STEC Screening GeneDisc, designed for
simultaneous detection of genes encoding Shiga toxins 1 and 2
(stx1 and stx2), intimin (eae), the E. coli O157 antigen (rfbE), and
Salmonella spp.-specific genes;
(2) the EHEC Identification GeneDisc Pack, designed for simultaneous
detection of O-group-associated genes of enterohemorrhagic E.
coli (EHEC) O26, O103, O111, O145 and the flagellar H7 (fliC H7)
gene present in EHEC O157:H7.
STEC O157
Salmonella
mTSB+n
↓
VIDAS-ICE
↓
CT-SMAC & CT-CHROM
BPW
↓
MSRV
↓
BGA & MLCB
Results
Only results obtained for the detection of STEC in animal feces are shown.
So far, only 16 meat samples have been analysed; 5 enriched in both
mTSB+n and BPW, 9 in only mTSB+n, and 2 in only BPW.
PCR efficiency (%) (pure culture)
Screening Disc
Lightcycler 2.0
137
106
119
98 & 107
132
101
O157
stx1 & stx2
eae
Conclusion
For the detection of STEC O157 in animal feces the VIDAS ICE procedure as
performed in this study is recommended above screening for the E. coli O157 antigen
encoding gene with the PCR systems tested.
Acceptable PCR efficiency: 80-120%
Comparing the results of the different combinations of enrichment and PCR systems,
systematically more fecal samples were identified as being stx-positive when enriched
in mTSB+n and subsequently screened with the Lightcycler 2.0 (Binomial test, P<0.05).
The highest prevalence rates for O26, O103 and O145 were obtained when samples
were enriched in BPW and screened with the Lightcycler 2.0 and for O111 after
enrichment in mTSB+n prior to screening with the Lightcycler 2.0. However, the
differences in prevalence rates obtained with the different methods of enrichment
combined with the 2 PCR systems were not statistically significant (P≥0.05).
Target
O157a
stx1 & stx2a
eaea
O26i
O103i
O111i
O145i
Relative sensitivity (%) of the O157-PCRs
compared with the VIDAS ICE procedure
Screening Disc
Lightcycler 2.0
76.5
76.5
41.2
70.6
mTSB+n
BPW
With the reference method, VIDAS ICE, STEC O157 were isolated from 17
out of 83 animals fecal samples, after enrichment in TSB+n.
Prevalence (%)
Relative sensitivity (%)
GeneDisc Cycler / Lightcycler 2.0
GeneDisc Cycler as alternative Lightcycler 2.0 as alternative
Lightcycler 2.0 as reference GeneDisc Cycler as reference
mTSB+n
BPW
38.6 / 47.0b
61.4d / 88.0d,e,f
92.8 / 94.0
54.3 / 52.2
23.9 / 26.1
6.5 / 10.9
43.5 / 52.2
26.5b,c / 53.0c
61.4e,g / 84.3f,g
84.3h / 97.6h
58.7 / 60.9
19.6 / 32.6
8.7 / 8.7
52.2 / 56.5
a n=83; b-h prevalence rates with the same superscript are
statistically significantly different (P<0.05); i n=46
Target
O157a
stx1 & stx2a
eaea
O26b
O103b
O111b
O145b
a n=83; b n=46
Relative accuracy (%)
mTSB+n
BPW
mTSB+n
BPW
76.9 / 93.8
68.5 / 98.0
97.4 / 98.7
91.7 / 88.0
75.0 / 81.8
60.0 / 100.0
83.3 / 100.0
47.7 / 95.5
72.9 / 100.0
86.4 / 100.0
92.9 / 96.3
53.3 / 88.9
100.0 / 100.0
88.5 / 95.8
86.7
71.1
96.4
89.1
89.1
95.7
91.3
71.1
77.1
86.7
93.5
82.6
100.0
91.3