Journal of microbiology and biotechnology, 2014, 01, 862─868

Development of a One-Step PCR Assay wth Nine Primer Pairs for the Detection of Five Diarrheagenic Escherichia coli Types

Kyung-Hwan Oh, Soo-Bok Kim, Mi-sun Park, Seung-Hak Cho

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 (MP-PCR) 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 (http://bioinfo.genotoul.fr/multalin/multalin.html). 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 (LODs) 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 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.

• ISBN or ISSN: 1017-7825

• 본 연구는 질병관리본부 연구개발과제(과제번호 2011-N41001-00) 연구비를 지원받아 수행되었습니다.
• This research was supported by a fund(code 2011-N41001-00) by Research of Korea Centers for Disease Control and Prevention.