Development of a One-Step PCR Assay with Nine Primer Pairs for the Detection of Five Diarrheagenic Escherichia coli Types
2
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
Certain Escherichia coli (E. coli) strains have the ability to cause diarrheal disease. Five types of diarrheagenic E. coli have been identified, including EHEC, ETEC, EPEC, EAEC, and EIEC. To detect these five diarrheagenic types rapidly, we developed a one-step multiplex PCR (MPPCR) assay using nine primer pairs to amplify nine virulence genes specific to the different virotypes, with each group being represented (i.e., stx1 and stx2 for EHEC, lt, sth, and stp for ETEC, eaeA and bfpA for EPEC, aggR for EAEC, and ipaH for EIEC). The PCR primers were constructed using MultAlin. The sensitivity and specificity of the constructed multiplex PCR primers were measured using DNA isolated from diarrheagenic E. coli strains representing each group. The limits of detection were as follows: 5 × 101 CFU/ml for EHEC, 5 × 103 CFU/ml for ETEC expressing lt and sth, 5 × 104 CFU/ml for ETEC expressing stp, 5 × 102 CFU/ml for EPEC, 5 × 104 CFU/ml for EAEC, and 5 × 102 CFU/ml for EIEC. To confirm the specificity, C. jejuni, C. perfringens, S. Typhimurium, V. parahaemolyticus, L. monocytogenes, Y. enterocolitica, B. cereus, and S. aureus were used as negative controls, and no amplification was obtained for these. Moreover, this kit was validated using 100 fecal samples from patients with diarrhea and 150 diarrheagenic E. coli strains isolated in Korea. In conclusion, the multiplex PCR assay developed in this study is very useful for the rapid and specific detection of five diarrheagenic E. coli types. This single-step assay will be useful as a rapid and economical method, as it reduces the cost and time required for the identification of diarrheagenic E. coli.
Abstract
Certain Escherichia coli (E. coli) strains have the ability to cause diarrheal disease. Five types of diarrheagenic E. coli have been identified, including EHEC, ETEC, EPEC, EAEC, and EIEC. To detect these five diarrheagenic types rapidly, we developed a one-step multiplex PCR (MPPCR) assay using nine primer pairs to amplify nine virulence genes specific to the different virotypes, with each group being represented (i.e., stx1 and stx2 for EHEC, lt, sth, and stp for ETEC, eaeA and bfpA for EPEC, aggR for EAEC, and ipaH for EIEC). The PCR primers were constructed using MultAlin. The sensitivity and specificity of the constructed multiplex PCR primers were measured using DNA isolated from diarrheagenic E. coli strains representing each group. The limits of detection were as follows: 5 × 101 CFU/ml for EHEC, 5 × 103 CFU/ml for ETEC expressing lt and sth, 5 × 104 CFU/ml for ETEC expressing stp, 5 × 102 CFU/ml for EPEC, 5 × 104 CFU/ml for EAEC, and 5 × 102 CFU/ml for EIEC. To confirm the specificity, C. jejuni, C. perfringens, S. Typhimurium, V. parahaemolyticus, L. monocytogenes, Y. enterocolitica, B. cereus, and S. aureus were used as negative controls, and no amplification was obtained for these. Moreover, this kit was validated using 100 fecal samples from patients with diarrhea and 150 diarrheagenic E. coli strains isolated in Korea. In conclusion, the multiplex PCR assay developed in this study is very useful for the rapid and specific detection of five diarrheagenic E. coli types. This single-step assay will be useful as a rapid and economical method, as it reduces the cost and time required for the identification of diarrheagenic E. coli.
참고문헌 (23)
- Bernier C, "Identification of an aggregative adhesion fimbria (AAF) type III-encoding operon in enteroaggregative Escherichia coli as a sensitive probe for detecting the AAF-encoding operon family", Infect. Immun, 70, pp.4302~4311 (2002)
- Botteldoorn N, "Detection and characterization of verotoxigenic Escherichia coli by VTEC", EHEC multiplex PCR in porcine faeces and pig carcass swabs, 2003, pp.154, 97~104
- Clarke SC., "Diarrhoeagenic Escherichia coli: an emerging problem?", Diagn. Microbiol. Infect. Dis, 41, pp.93~99 (2001)
- Corpet F., "Multiple sequence alignment with hierarchical clustering", Nucl. Acids Res, 16, pp.10881~10890 (1988)
- Fujuoka M, "Direct detection of diarrheagenic Escherichia coli in patient stool specimens by developed multiplex PCRs for the establishment of surveillance system of diarrheagenic Escherichia coli", Jpn. J. Med. Technol, 8, pp.1041~1046 (2008)
- Fujioka M, "A novel single-step multiplex polymerase chain reaction assay for the detection of diarrheagenic Escherichia coli", J. Microbiol. Methods, 92, pp.289~292 (2013)
- Gomez-Duarte OG, "Detection of E. coli, Salmonella spp., Shigella spp., Yersinia entercolitica, Vibrio cholerae, and Campylobacter spp. enteropathogens by threereaction multplex PCR", Diagn. Microbiol. Infect. Dis, 63, pp.1~9 (2009)
- Kaper JB, "Pathogenic Escherichia coli", Nat. Rev. Microbiol, 2, pp.123~140 (2004)
- Lang AL, "Multiplex PCR for detection of the heat-labile toxin gene and Shiga-like toxin I and II genes in Escherichia coli isolated from natural waters", Appl. Environ. Microbiol, 60, pp.3145~3149 (1994)
- Lasaro MA, "Genetic diversity of heat-labile toxin expressd by enterotoxigenic Escherichia coli strains isolated from humans", J. Bacteriol, 190, pp.2400~2410 (2008)
- Nataro JP, "Diarrheagenic Escherichia coli", Clin. Microbiol. Rev, 11, pp.142~201 (1998)
- Nataro JP, "AggR, a transcriptional activator of aggregative adherence factor I expression", J. Bacteriol, 176, pp.4691~4699 (1994)
- Osek J., "Development of a multiplex PCR approach for the identification of Shiga toxin-producing Escherichia coli strains and their major virulence factor genes", J. Appl. Microbiol, 95, pp.1217~1225 (2003)
- Pass MA, "Multiplex PCR for identification of Escherichia coli virulence genes", J. Clin. Microbiol, 38, pp.2001~2004 (2000)
- Rappelli P, "Development of a set of multiplex PCR assays for the simultaneous identification of enterotoxigenic, enteropathogenic, enterohemorrhagic and enteroinvasive Escherichia coli", Microbiologica, 24, pp.77~83 (2001)
- Savarino SJ, "Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another family of E. coli heat-stable toxin", Proc. Natl. Acad. Sci. USA, 90, pp.3093~3097 (1993)
- Sethabutr O, "Detection of PCR products of the ipaH gene from Shigella and enteroinvasive Escherichia coli by enzyme linked immunosorbent assay", Diagn. Microbiol. Infect. Dis, 37, pp.11~16 (2000)
- Sheikh J, "A novel dispersin protein in enteroaggregative Escherichia coli", J. Clin. Investig, 110, pp.1329~1337 (2002)
- Sjöling Å, "Comparative analyses of phenotypic and genotypic methods for detection of enterotoxigenic Escherichia coli toxins and colonization factors", J. Clin. Microbiol, 45, pp.3295~3301 (2007)
- Tarr PI, "Shiga-toxinproducing Escherichia coli and haemolytic uraemic syndrome", Lancet, 365, pp.1073~1086 (2005)
- Tobias J, "Simple and rapid multiplex PCR for identification of the main human diarrheagenic Escherichia coli", Microbiol. Res, 167, pp.564~570 (2012)
- Trabulsi LR, "Typical and atypical enteropathogenic Escherichia coli", Emerg. Infect. Dis, 8, pp.508~513 (2002)
- Watterworth L, "Multiplex PCR-DNA probe assay for the detection of pathogenic Escherichia coli", J. Microbiol. Methods, 60, pp.93~105 (2005)