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Journal of Microbiological Methods 55 (2003) 383 – 392 www.elsevier.com/locate/jmicmeth Comparison of different PCR tests for detecting Shiga toxin-producing Escherichia coli O157 and development of an ELISA-PCR assay for specific identification of the bacteria Patrick Fach *, Sylvie Perelle, Joël Grout, Francßoise Dilasser Agence Francßaise de Sécurité Sanitaire des Aliments (AFSSA), Laboratoire d’Etudes et de Recherches sur l’Hygiène et la Qualité des Aliments, Unité: Atelier de Biotechnologie, 1-5 rue de Belfort, 94700 Maisons-Alfort, France Received 3 January 2003; received in revised form 29 April 2003; accepted 28 May 2003 Abstract In an attempt to develop a standard for ELISA – PCR detection of Shiga toxin producing Escherichia coli (STEC) O157, six published PCR tests were tested in a comparative study on a panel of 277 bacterial strains isolated from foods, animals and humans. These tests were based on the detection of the genes rfbE [J. Clin. Microbiol. 36 (1998) 1801] and rfbB [Appl. Environ. Microbiol. 65 (1999) 2954], the 3Vend of the eae gene [Epidemiol. Infect. 112 (1994) 449], the region immediately flanking the 5V end of the eae gene [Int. J. Food. Microbiol. 32 (1996) 103], the flicH7 gene [J. Clin. Microbiol. 35 (1997) 656], or a part of the recently described 2634-bp Small Inserted Locus (SILO157 locus) of STEC O157 [J. Appl. Microbiol. 93 (2002) 250]. Unlike the other PCR assays, those amplifying the rfb sequences were unable to distinguish toxigenic from nontoxigenic O157. These assays were relatively specific to STEC O157, giving essentially a cross reaction with clonally related E. coli O55 and to a lesser extent with E. coli O145, O125, O126. They also detected the Shiga toxin (stx)-negative derivatives of STEC O157. Based on these results, an ELISA-PCR assay consisting of the solution hybridization of amplicons with two probes that ensured the specificity of the amplification was developed. The ELISA-PCR assay, which used an internal control (IC) of inhibition, was able to detect 1 to 10 copies of STEC O157 in the PCR tube. Adaptation of PCR into ELISA-PCR assay format facilitates specific and sensitive detection of PCR amplification products and constitutes a method of choice for screening STEC O157. D 2003 Elsevier B.V. All rights reserved. Keywords: Enterohemorrhagic Escherichia coli; O157; ELISA-PCR 1. Introduction Since its recognition in 1982, enterohemorrhagic Escherichia coli O157 (EHEC O157) has been recognized as an important human pathogen predominantly * Corresponding author. Tel.: +33-1-43-76-30-99; fax: +33-143-76-26-30. E-mail address: [email protected] (P. Fach). associated with hemorrhagic colitis and the more severe complications of hemolytic uremic syndrome. It causes high morbidity and mortality especially among the young and elderly. In the last few years, EHEC O157 has been responsible for a number of well-publicized outbreaks in many parts of the world, and various methods have been developed to detect the bacteria. Several selective differentiating media, immunological assays, and DNA probes have been used. 0167-7012/03/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0167-7012(03)00172-6 384 P. Fach et al. / Journal of Microbiological Methods 55 (2003) 383–392 The cultural methods have exploited the biochemical characteristics of the O157:H7 strains, which do not produce h-glucuronidase and usually do not ferment D-sorbitol rapidly (Thompson et al., 1990; Nataro and Kaper, 1998). The agar medium most commonly used to isolate E. coli O157:H7 is sorbitol-MacConkey (SMAC) agar containing cefixime and tellurite. It supports stx-producing E. coli O157:H7 (STEC O157) and inhibits the growth of most of the other E. coli strains. The disadvantage of these culture methods is that they do not differentiate between toxigenic and stx-negative derivatives of STEC O157, found in patients, animals or subcultures. In addition, they fail to detect some atypical strains of STEC O157 that may occur (Gunzer et al., 1992). Most of the immunological assays available for identification of the bacteria detect either O and H antigens or Shiga-like toxin. However, cross-reactivity with organisms other than E. coli O157 has been reported in immunoassays. Anti-O157 sera may cross-react with Escherichia hermanii, Salmonella O group N, Hafnia alvei, Yersinia enterocolitica or to a lesser extent with Citrobacter freundii (Bettelheim et al., 1993; Borczyk et al., 1987; Chart et al., 1992; Nataro and Kaper, 1998; Shimada et al., 1992) and the suspected colonies have to be confirmed as E. coli by biochemical identification. To achieve accurate and rapid epidemiological investigation of outbreaks, it is important to be able to fully distinguish EHEC O157 from other bacteria and from bacteria of serogroup O157 that are not pathogenic. Nucleic acid amplification technologies offer the potential for improved detection of EHEC O157 in the environment providing greater sensitivity and dramatically speeding up detection to improve the management of outbreaks through more-rapid confirmation of the vehicle of infection. A number of PCR methods have been reported for the detection of EHEC O157. Usually, the assays involve multiplex PCR targeting different genes that are not specific to EHEC O157 alone (Cebula et al., 1995; Deng and Fratamico, 1996; Fratamico et al., 1995; Gannon et al., 1997; Nagano et al., 1998; Paton and Paton, 1998). These tests are suited to the identification of isolated strains of bacteria and have been shown to be specific and sensitive for the examination of complex matrices such as foods and stools (Sharma and Dean-Nystrom, 2003). However, in a mixture of bacteria, the genetic profile obtained by multiplex PCR may arise from more than one strain, hindering interpretation. Ambiguity arises when both stx and rfbO157 genes are detected in a sample containing different strains of E. coli. In this situation, it is not possible to ensure that the signal obtained in multiplex PCR is displayed by STEC O157. The mixture may contain non-Shiga toxin-producing E. coli O157 (EC O157) together with STEC from another serogroup. The additive effect of different strains in multiplex PCR described by Deng and Fratamico (1996) underlines the need to find a single specific DNA sequence to identify EHEC O157. Moreover, one of the major drawbacks of the published PCR-based approaches for detection of STEC O157 is lack of internal amplification control (IAC) which is required in order to monitor false negatives that may be caused by PCR inhibitors. Among the more recent PCR techniques used for detecting STEC O157, real-time PCR using either SYBR Green I or TaqMan assay is a powerful technique. Real-time PCR techniques provide results available immediately after completion of the amplification reaction, with no need for any further processing of the samples and without opening the test tubes, greatly reducing the risk of carry-over contamination. Another advantage of real-time PCR methods is that they do not need ethidium bromide, which is subject to strict and constraining regulations in many countries, is increasingly restricted in the food industry and less and less common in routine laboratory use. In the present study, we evaluated on the same large collection of bacteria six different nonmultiplexed PCR systems used for detecting EHEC O157. Based on these results, an ELISA-PCR assay including a solution hybridization step was developed for confirmation of PCR amplification products. Furthermore, an internal control (IC) of inhibition was designed in order to increase the reliability of the diagnostic ELISA-PCR assay. Comparing our data with those of the literature, the ELISA-PCR test yielded results comparable to those obtained in many other published E. coli O157 PCR detection systems. There is no evidence for significant differences in terms of sensitivity and efficiency compared with the real-time PCR TaqMan assays described in the literature for E. coli O157 (Oberst et al., 1998; Sharma, 2002). The threshold sensitivity of both ELISA-PCR and real-time PCR systems is approximately 102 CFU P. Fach et al. / Journal of Microbiological Methods 55 (2003) 383–392 ml 1, which corresponds to less than 10 cells in the PCR tube. 2. Materials and methods 2.1. Bacterial strains and culture conditions A total of 277 strains were used for the specificity study. They comprised 67 strains of Shiga toxinproducing E. coli O157 (STEC O157), 93 non-Shiga toxin-producing E. coli O157 (EC O157), 91 STEC of other serogroups: O3 (2), O4 (1), O5 (7), O6 (9), O8 (1), O15 (1), O22 (2), O26 (8), O45 (1), O53 (2), O55 (1), O76 (3), O77 (3), O79 (1), O86 (1), O88 (1), O91 (6), O103 (7), O110 (2), O111 (11), O113 (3), O117 (2), O118 (2), O125 (1), O126 (1), O128 (1), O136 (2), O138 (1), O139 (2), O141 (1), O145 (4), O147 (1), 12 non-stx E. coli: O26 (2), O55 (3), O103 (1), O111 (2), O125 (1), O126 (1), O127 (1), O128 (1); and 18 strains of other bacterial species. The strains were obtained from several laboratories (see Acknowledgments) or isolated in our laboratory (Fach et al., 2001). All the strains were grown in tryptone soy broth (TSB) and incubated at 37 jC overnight. Enumeration of STEC O157 strains used to determine the threshold sensitivity of the ELISA-PCR was performed by double plating as previously described (Fach et al., 2001). 2.2. DNA extraction One milliliter of pure culture of bacteria was placed in an Eppendorf tube, centrifuged at 12 000 g for 2 min, and the supernatant decanted. The pellet was washed in 1 ml PBS, pH 7.5 and mixed with 200 Al of InstaGenek Matrix (Bio-Rad, Marnes-la-Coquette, France). Bacterial DNA was released by heating the sample for 15 min at 56 jC and 8 min at 100 jC. After vortexing and centrifugation at 14 000 g for 2 min, 10 Al of the supernatant was then used in the PCR reactions. 2.3. Characterization of isolated bacteria Bacterial strains were identified by the API-20 test (bioMérieux, Marcy-l’Etoile, France). Overnight cultures of E. coli were transferred to SMAC plates 385 containing sorbitol MacConkey agar (Difco) and incubated for 18 to 24 h at 37 jC to identify sorbitolfermenting activity of clones. The O157 phenotype of strains was examined by the E. coli O157 Latex Test from Oxoid, according to the manufacturer’s instructions (Oxoid, Dardilly, France). Other serogroups were characterized at the Danish Veterinary Laboratory (Copenhagen, Denmark) by O-typing, using rabbit E. coli antisera. The Shiga toxin type was identified by the Vero cell assay (VCA) as described below, and by specific PCR of the stx1 and stx2 genes as described by Pollard et al. (1990a,b). The presence of additional virulence factors responsible for attaching/ effacing lesions (eaeA gene), enterohemolysin (hlyA gene) and catalase-peroxidase (katP gene) production was determined by previously described PCR (Brunder et al., 1996; Sandhu et al., 1996; Schmidt et al., 1995). 2.4. Vero cell assay Detection of Shiga toxin-producing bacteria was performed using a Vero cell assay (VCA) technique, as described previously in the literature (Konowalchuk et al., 1977; Strockbine et al., 1986). Briefly, 1 ml of the enrichment culture was centrifuged and the pellet was treated with 2000 U ml 1 of polymyxin B (Sigma, France) at 37 jC for 45 min. After centrifugation and filtration of the supernatant through a 0.22-Am membrane filter, the cytotoxicity was evaluated on Vero cells. Cell viability was observed for 4 days. When cytotoxicity was found, the Shiga toxin production was confirmed by a seroneutralization test using the reference monoclonal antibodies 13C4 (ATCC No. CRL 1794) (Strockbine et al., 1985) and 11F11 (ATCC No. CRL 1908) (Perera et al., 1988). 2.5. PCR assays for detecting Shiga toxin-producing E. coli O157 The six PCR assays amplifying the genes rfbE (Desmarchelier et al., 1998) and rfbB (Maurer et al., 1999), the 3Vend of the eae gene (Louie et al., 1994), the region immediately flanking the 5Vend of the eae gene (Meng et al., 1996), the flicH7 gene (Gannon et al., 1997), or a part of the recently described SILO157 locus of STEC O157 (Perelle et al., 2002) were performed in the GeneAmp PCR System 9700 (Ap- 386 P. Fach et al. / Journal of Microbiological Methods 55 (2003) 383–392 plied Biosystems, Courtaboeuf, France) in a final volume of 50 Al containing 200 AM of each dNTP, 600 nM of each primer, 5 Al of the GeneAmpR 10 PCR buffer (Applied Biosystems), 2.5 U of AmpliTaq Goldk (Applied Biosystems) and 10 Al of the extracted DNA. The PCR cycling conditions used with the six sets of primers (Table 1) were as follows: an initial denaturation at 94 jC for 10 min followed by 35 cycles, each consisting of 30 s at 94 jC, 20 s at the annealing temperature (see Table 1), and 30 s at 72 jC. After a final elongation step at 72 jC for 10 min, the PCR products were electrophoresed on 1 – 2% agarose gels containing ethidium bromide in 1 TBE buffer. SF6 primer binding regions was cloned in the pMOSBlue plasmid and formed the synthetic IC. Simultaneous amplification of these two different DNA fragments flanked by the same primer sites results in a competitive PCR, depending on the molar ratio of those DNA fragments. The number of IC copies in each PCR tube was limited to 10 copies to ensure competition advantages for STEC O157. IC was developed to monitor potential PCR inhibitors and ensure successful amplification. Owing to the competition of the two PCR reactions, if the SILO157 locus was amplified but not the IC, then it was assumed that STEC O157 DNA was present in a proportionally greater amount. If neither the IC nor the SILO157 locus was amplified, then it was assumed that inhibition of the PCR had occurred and the test for that sample was not valid. After amplification, the PCR products were detected in a sandwich hybridization assay as previously described (Fach et al., 2001). This step was performed in parallel on two microtiter plates; one for the specific detection of the SILO157 locus and one for the IC detection. The set of internal capture and detection probes for detection of the SILO157 locus was 388S oligonucleotide 5V-end labeled with biotin and 388R oligonucleotide 3V-end labeled with digoxigenin (Table 1). The set of internal capture and detection probes for detection of IC was CatCap oligonucleotide 5Vend-labeled with biotin and CatRev oligonucleotide 2.6. ELISA-PCR assay to detect the SILO157 locus Amplification reactions with the RJD3 and SF6 primers were performed as described above, using 10 copies of a synthetic IC in each reaction. IC was synthesized as previously described (Fach et al., 2001), in a one-step PCR reaction with primers bearing 5Voverhanging ends identical to the published primers RJD3 and SF6 used in STEC O157 diagnostic reaction, and 3’ ends complementary to a DNA sequence of the Cm gene from Tn9. This feature allowed RJD3 and SF6 primers to co-amplify the SILO157 locus from STEC O157 and the recombinant Cm gene sequence in the same reaction. The recombinant Cm gene sequence flanked by the RJD3 and Table 1 Sequence of primers and probes Primers and probes Sequence (5V– 3V) Annealing temperature Amplicon size Target gene Reference O157 A-F O157 A-R O157 P-F8 O157 P-R8 P1EH P2EH FLIC H7-F FLIC H7-R SZ I SZ II RJD3 SF6 388S 388R CatCap CatRev AAGATTGCGCTGAAGCCTTTG CATTGGCATCGTGTGGACAG CGTGATGATGTTGAGTTG AGATTGGTTGGCATTACTG AAGCGACTGAGGTCACT ACGCTGCTCACTAGATGT GCGCTGTCGAGTTCTATCGAGC CAACGGTGACTTTATCGCCATTCC CCATAATCATTTTATTTAGAGGGA GAGAAATAAATTATATTAATAGATCGGA TTAAAACCGGTGACGTGATGATGGTG CGCAGAAATACCGGCTTTAAGTACC AGCGGGAGCGGGAACCTTCAACGGTGAATCT GTGTCTGTCGGAACATCATTTAATGCCGGAA TCGCAAGATGTGGCGTGTTACGGTGAAAAC CAAGGCGACAAGGTGCTGATGCCGCTGGCG 66 jC 497 bp 55 jC 420 bp 55 jC 476 bp 65 jC 625 bp 60 jC 632 bp 71 jC 125 bp – – – – – – – – rfbEO157 rfbEO157 rfbBO157 rfbBO157 eaeAO157 eaeAO157 fliCH7 fliCH7 OrfuLEE O157 OrfuLEE O157 SILO157 SILO157 SILO157 SILO157 Cm Cm Desmarchelier et al., 1998 Desmarchelier et al., 1998 Maurer et al., 1999 Maurer et al., 1999 Louie et al., 1994 Louie et al., 1994 Gannon et al., 1997 Gannon et al., 1997 Meng et al., 1996 Meng et al., 1996 Perelle et al., 2002 Perelle et al., 2002 This study This study Fach et al., 2001 Fach et al., 2001 P. Fach et al. / Journal of Microbiological Methods 55 (2003) 383–392 3V-end-labeled with digoxigenin (Fach et al., 2001) (Table 1). Thus using two different sets of internal capture and detection probes in two distinct microtiter plates, it was possible to differentiate amplicons derived from the SILO157 locus from those derived from the IC. 2.7. Determination of the ELISA-PCR assay cut-off The RJD3-SF6 amplified fragment from the SILO157 locus was cloned into pMOSBlue vector using the pMOSBlue blunt ended cloning kit (Amersham Biosciences, Saclay, France). The resulting recombinant plasmid DNA was purified using QIAGENR plasmid kit (QIAGEN, Courtaboeuf, France) according to the manufacturer’s instructions. Its concentration was determined by fluorimetry and the copy number of the plasmid was calculated according to its size. The cloned sequence was used as positive control and to determine the detection limit (cut-off) of the ELISA-PCR. The threshold selection criterion determining positive versus negative samples was established by testing triplicates of serial 10-fold dilutions (106 to 1 copies ml 1) of the plasmid in ELISA-PCR. The mean values were calculated for each dilution and for the negative controls, and standard deviations were also determined. 3. Results and discussion 3.1. Comparison of PCR assays for detecting Shiga toxin-producing E. coli O157 Several simplex (nonmultiplex) PCR assays were compared for detecting STEC O157. Specificity of the PCR assays was evaluated with 277 bacterial strains isolated from foods, animals or humans and six different simplex PCR assays amplifying the genes rfbE and rfbB, the 3Vend of the eae gene, the region immediately flanking the 5Vend of the eae gene, the flicH7 gene, or a part of the SILO157 locus of STEC O157. Data showed that the PCR assays amplifying the rfb sequences indifferently detected all the 67 strains of toxigenic E. coli O157 and the 93 strains of nontoxigenic E. coli O157 that were isolated essentially from animals and foods. These nontoxigenic E. coli O157 frequently encountered in certain samples, such as bovine sam- 387 ples, were usually sorbitol-positive, had an H-type other than H7, and were negative for the eaeA, hlyA, katP EHEC virulence genes. Such strains, which have to be unequivocally distinguished from toxigenic E. coli O157, could be differentiated using the four other PCR systems tested. The stx-negative derivatives of STEC O157 (eight strains) that were sorbitol-negative and positive for the eaeA, hlyA, katP EHEC virulence genes could not be differentiated from toxigenic E. coli O157 and tested positive in the six PCR assays. These PCR systems, when evaluated on 34 serogroups of E. coli (including STEC and non-STEC), showed a high specificity for E. coli O157, giving cross reactions with only a few E. coli strains of STEC or EPEC belonging to other serogroups. Thus PCR tests amplifying the 3V end of the eae gene and the region immediately flanking the 5Vend of the eae gene detected strains of E. coli O55 and O145. The EPEC strains O125:H6 and O126:H6 also tested positive by amplification of the region immediately flanking the 5Vend of the eae gene. PCR assay targeting the SILO157 locus was found to be more specific, detecting toxigenic O157 and only clonally related E. coli O55. Amplification of the flicH7 gene of STEC O157 allowed the detection of toxigenic E. coli O157 of H7 type and strains of STEC or EPEC belonging to the O55:H7 serogroup while the nontoxigenic E. coli O157 of H types other than H7 such as HND, H19 or H45 were not detected. Thus the simplex PCR assays for detecting toxigenic E. coli O157 were relatively specific to STEC O157, essentially giving cross reactions with O55:H7 and to a lesser extent with O125:H6, O126:H6 and O145 (Tables 2 and 3). They also detected the stx-negative derivatives of STEC O157, occurring in patients, animals or subcultures (Table 2). However, they did not detect the 14 strains of other bacteria such as C. freundii, Enterobacter sakasaki, H. alvei and Salmonella enterica group O301 which cross react with the E. coli O157 Latex Test (Table 4). 3.2. ELISA-PCR test with cloned sequence of the SILO157 locus and pure culture of bacteria The threshold sensitivity of the ELISA-PCR assay was determined by testing 10 Al of 10-fold serial dilutions of the cloned sequence of the SILO157 (data not shown). DNA plasmid concentrations of 106 and 105 copies ml 1 (104 and 103 copies in the PCR tube) 388 P. Fach et al. / Journal of Microbiological Methods 55 (2003) 383–392 Table 2 Examination of E. coli O157 isolates by PCR Bacteria Number tested Shiga toxin-producing E. coli O157 (STEC O157:H7, NSF, Stx1 positive O157:H7, NSF, Stx2 positive O157:H7, NSF, Stx1 and Stx2 positive O157:H-, NSF, Stx2 positive O157:H-, SF, Stx2 positive O157:H-, NSF, Stx1 and Stx2 positive O157:Hnd, NSF, Stx2 positive O157) 3a 16a 28a 4a 1a 2a 13a Non-Shiga toxin-producing E. coli O157 (EC O157) O157:Hnd, SF, Stx negative 76b O157:Hnd, NSF, Stx negative 4b O157:H7, NSF, Stx negative 4a O157:Hnd, NSF, Stx negative 4a O157:H19, SF, Stx negative 2b O157:H45, SF, Stx negative 1c O157:Hnd, SF, Stx negative 2b,d PCR assays rfbEO157 rfbBO157 eaeAO157 fliCH7 orfULEE + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + O157 SILO157 NSF: non-sorbitol fermenting, SF: sorbitol fermenting, ND: not determined. a Positive for the EHEC virulence eaeA, hlyA, katP genes. b Negative for the EHEC virulence eaeA, hlyA, katP genes. c Positive for the EHEC virulence eaeA gene. d Positive for the ETEC virulence ST. gave an absorbance value of 4.0, and DNA plasmid concentrations ranging from 104 to 103 copies ml 1 (102 – 10 copies in the PCR tube) gave absorbance values between 3.620 and 4.0. Concentrations of 102 copies ml 1 (approximately one copy in the PCR tube) usually yielded absorbance values lower than 0.03, except for one sample which was 0.140. For concentrations under 10 copies ml 1 (less than one copy in the PCR tube) absorbance values were lower than 0.03. Mean values and standard deviation of PCR Table 3 Examination of non-O157 E. coli isolates by PCR Bacteria Number tested PCR assays rfbEO157 rfbBO157 E. coli giving a negative reaction with the O157 RPLA test O55:H7, SF, Stx2 positive 1a,b O55:H7, SF, Stx negative 3a,b O125:H6, SF, Stx negative 1a,b O126:H6, NSF, Stx negative 1a,b O145:H-, SF, Stx1 positive 1c O145:H-, SF, Stx2 positive 1d O145:H-, SF, Stx negative 1c O145:Hnd, SF, Stx2 positive 1d Other serogroups of E. coli 93 (see Material and methods) NSF: non-sorbitol fermenting, SF: sorbitol fermenting, ND: not determined. a Negative for the EHEC virulence hlyA, katP genes. b Positive for the EHEC virulence eaeA gene. c Positive for the EHEC virulence eaeA, hlyA, katP genes. d Positive for the EHEC virulence eaeA, hlyA genes. eaeAO157 fliCH7 orfULEE + + + + + + + + + + + + + + + + O157 SILO157 + + P. Fach et al. / Journal of Microbiological Methods 55 (2003) 383–392 389 Table 4 Examination of other bacterial species isolates by PCR Bacteria Number tested PCR assays rfbEO157 rfbBO157 eaeAO157 fliCH7 orfULEE O157 SILO157 Bacteria other than E. coli giving a positive reaction with the O157 RPLA test Citrobacter freundii, NSF, Stx negative 1 Enterobacter sakasaki, SF, Stx negative 1 Hafnia alvei, NSF, Stx negative 4 Salmonella enterica group O301 (group N) S. aqua, SF, Stx negative 1 S. aschersleben, SF, Stx negative 1 S. bodjonegoro, SF, Stx negative 1 S. landau, SF, Stx negative 1 S. morehead, SF, Stx negative 1 S. ramatgan, SF, Stx negative 1 S. soerenga, SF, Stx negative 1 S. urbana, SF, Stx negative 1 Other Stx negative species of bacteria Pseudomonas aeruginosa, Pseudomonas fluorescens 4 Table 5 ELISA-PCR with pure culture of STEC O157 STEC O157 strains VCA Numeration (CFU ml Theoretical O157:H7 (ATCC 43890) Stx1 O157:H(E-32511) Stx2 O157:H7 (ATCC 43895) Stx1, Stx2 0 1 10 102 103 104 105 106 0 1 10 102 103 104 105 106 0 1 10 102 103 104 105 106 1 ) Estimated by plating Estimated number of copies in the PCR tube 0 2.4 24 2.4 102 2.4 103 2.4 104 2.4 105 2.4 106 0 3.1 31 3.1 102 3.1 103 3.1 104 3.1 105 3.1 106 0 3 30 3 102 3 103 3 104 3 105 3 106 0 0.12 1.2 12 1.2 102 1.2 103 1.2 104 1.2 105 0 0.15 1.55 15.5 1.55 102 1.55 103 1.55 104 1.55 105 0 0.15 1.5 15 1.5 102 1.5 103 1.5 104 1.5 105 ELISA-PCR SILO157 IC Test 1 Test 2 Test 1 Test 2 0.001 3.565 4.000 4.000 4.000 4.000 4.000 4.000 0.004 0.001 4.000 4.000 4.000 4.000 4.000 4.000 0.011 0.005 3.677 4.000 4.000 4.000 4.000 4.000 0.005 4.000 4.000 4.000 4.000 4.000 4.000 4.000 0.008 4.000 4.000 4.000 4.000 4.000 4.000 4.000 0.004 0.003 3.737 4.000 4.000 4.000 4.000 4.000 3.699 3.694 3.428 3.391 1.217 0.585 0.029 0.028 4.000 3.827 3.491 3.078 1.457 0.468 0.049 0.033 3.786 3.778 3.628 3.434 2.143 0.343 0.078 0.028 3.794 3.697 3.589 3.501 2.506 0.497 0.029 0.025 4.000 3.630 3.607 3.582 3.349 2.399 0.074 0.012 4.000 4.000 3.796 3.516 3.065 0.107 0.058 0.025 390 P. Fach et al. / Journal of Microbiological Methods 55 (2003) 383–392 negative controls (sterile water) were also measured, and the cut-off of the test was established on the basis of mean values plus three standard deviations. Accordingly, absorbance values greater than or equal to 0.100 were considered positive, and values lower than 0.100 negative. The sensitivity of the ELISA-PCR was then evaluated with pure cultures of STEC O157 10-fold diluted from approximately 106 to 1 CFU ml 1 in buffered peptone water. The results indicated absorbance values greater than the cut-off for dilutions of approximately 24– 30 CFU ml 1 and above while under these dilutions, i.e. for samples theoretically containing less than one copy in the PCR tube, alternatively negative and positive results were observed (Table 5). As expected for high dilution levels, the copy number of target per PCR tube may vary from 0 to 10. In this situation, results of the assay are randomly positive and negative. STEC O157 and IC were co-amplified with one common set of primers, in the same conditions, and in the same PCR tube so that simultaneous amplification sometimes resulted in the inhibition of the IC, depending on the molar ratio of STEC O157 and IC. The number of IC copies in each PCR tube was limited to 10 to ensure competition advantages for STEC O157. IC was never detected in samples containing proportionally greater amounts of STEC O157. For samples containing more than 1.2 104 STEC O157 in the PCR tube, IC was unable to compete for primers and the IC signal was negative. As expected, IC was detected in all STEC O157 negative samples. 4. Conclusion Using E. coli O157 immunoassays, a few bacterial cells can be detected following enrichment in selective medium and subsequent isolation by immunomagnetic separation. However, these tests are prone to crossreactivity of O antisera with organisms other than E. coli O157 (Bettelheim et al., 1993; Borczyk et al., 1987; Chart et al., 1992; Nataro and Kaper, 1998; Shimada et al., 1992). Also, O157 has been reported to cross-react with 12 O-antigens of other E. coli Oserogroups (Aleksic et al., 1992). Thus reliable detection of STEC O157 needs confirmation of the suspected colonies by biochemical identification and Stx demonstration using either Vero cell assay, enzymelinked immunosorbent assay (ELISA) kits or PCR based on the stx genes. Multiplex PCR assays have been developed for identification of suspected clones isolated from water, food, or fecal samples (Cebula et al., 1995; Deng and Fratamico, 1996; Fratamico et al., 1995; Gannon et al., 1997; Nagano et al., 1998; Paton and Paton, 1998). Usually, they are based on detection of both stx and rfb or flicH7 genes and offer a rapid and reliable way to test isolated clones. However, in a mixture of bacteria they are sometimes difficult to manage because the genetic profile obtained by multiplex PCR may arise from more than one strain, hindering interpretation. When both stx and rfbO157 genes are detected by multiplex PCR in a mixture of bacteria, it is not possible to ensure that the signal is displayed by STEC O157. The mixture may contain non-Shiga toxin-producing E. coli O157 (EC O157) together with STEC from another serogroup. In that case, only isolation of E. coli clones and individual testing of the isolates by PCR can demonstrate the presence or absence of STEC O157 in the sample. Thus for rapid screening of these samples there was a need to select one simplex (nonmultiplex) PCR assay based on a single maximally specific DNA sequence identifying STEC O157. In this study, we compared, on the same large collection of bacteria, different nonmultiplexed PCR systems used for detecting EHEC O157 and chose the most appropriate system to adapt an ELISA-PCR assay. An IC was also included in this test to monitor false negative samples. The ELISAPCR assay consists of the solution hybridization of amplicons with two probes that ensure the specificity of the amplification. The first one is used as a capture probe and the second as a detection probe. The capture probe is labeled with biotin and is bound to streptavidin-coated microtiter plates, while the detection probe is labeled with digoxigenin. After alkaline denaturing of PCR products, hybridization occurs in parallel on two microtiter plates: one for the specific detection of STEC O157 and one for the internal control (IC) detection. Thus by using two different sets of internal capture and detection probes in two separate microtiter plates, it is possible to differentiate amplicons derived from STEC O157 from those derived from the IC. Similarly to the ELISA technology, hybrids are detected using a peroxidase anti-digoxigenin conjugate. The final enzymatic reaction of the test gives a P. Fach et al. / Journal of Microbiological Methods 55 (2003) 383–392 colorimetric signal measured by spectrophotometry. The ELISA-PCR test proved specific and highly sensitive; a positive signal was detected in samples containing less than 10 genomes of STEC O157 in the PCR tubes. Comparing our data with those of the literature, the ELISA-PCR test gave results comparable to those obtained in many other published E. coli O157 PCR detection systems. There is no evidence for significant differences in terms of sensitivity and efficiency compared with the real-time PCR TaqMan assays described in the literature for E. coli O157 (Oberst et al., 1998; Sharma, 2002). This method would constitute a valuable PCR screening method to detect STEC O157 in complex matrices like foods or stools. Experiments are in progress to evaluate it on naturally contaminated samples. However, as the number of STEC O157 positive samples is very low, such contamination was undetected in samples investigated at the moment in our laboratory. Acknowledgements We thank E. Oswald from INRA-ENVT, Toulouse, France, who provided most of the reference strains of STEC and the hybridoma 13C4 and 11F11, C. Lapeyre who produced and purified monoclonal antibodies, and M. Maire and C. Crucière from AFSSA for help in the Vero Cell Assay. We are grateful to K. Frydendahl from Danish Veterinary Laboratory, Copenhagen, Denmark, for help in O-typing of strains, and to the following for providing strains of bacteria: P. Pohl, NIDO-INRV, Brussels, Belgium; D. Piérard, AZVUB, Brussels, Belgium; B. China and J. Mainil, University of Liège, Belgium; G. Duffy, The National Food Centre, Dublin, Ireland; M. Doyle, University of Georgia, USA; D. Woodward and R. Caldeira, LCDC, Ottawa, Ontario, Canada; P. Fratamico, USDA, Philadelphia, PA, USA; A. Caprioli, Istituto Superiore di Sanità, Rome, Italy; J. Blanco, LREC, Universidad de Santiago de Compostela, Lugo, Spain; L. Beutin, RKI, Berlin, Germany; H. Schmidt, University of Würzburg, Germany; Y. Wasteson, The Norwegian School of Veterinary Science, Oslo, Norway; C. Vernozy-Rozand, ENVL, Marcy-l’Etoile, France; C. Doit, Hôpital Robert-Debré, Paris, France; B. Andral, AFSSA-Lyon, France and M. Bohnert, AFSSAPloufragan, France. 391 References Aleksic, S., Karch, H., Bockemuhl, J., 1992. 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