Oh Kyung-Hwan, Kim Soo-Bok, Park Mi-Sun, Cho Seung-Hak
Division of Enteric Diseases, Center for Infectious Diseases, Korea National Institute of Health, Osong 363-95, Republic of Korea.
J Microbiol Biotechnol. 2014 Jun 28;24(6):862-8. doi: 10.4014/jmb.1312.12031.
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 × 10(1) CFU/ml for EHEC, 5 × 10(3) CFU/ml for ETEC expressing lt and sth, 5 × 10(4) CFU/ml for ETEC expressing stp, 5 × 102 CFU/ml for EPEC, 5 × 10(4) CFU/ml for EAEC, and 5 × 10(2) 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.
某些大肠杆菌（E. coli）菌株具有引发腹泻疾病的能力。已鉴定出五种致泻性大肠杆菌，包括肠出血性大肠杆菌（EHEC）、肠产毒性大肠杆菌（ETEC）、肠致病性大肠杆菌（EPEC）、肠集聚性大肠杆菌（EAEC）和肠侵袭性大肠杆菌（EIEC）。为了快速检测这五种致泻性类型，我们开发了一种一步多重PCR（MPPCR）检测方法，使用九对引物来扩增不同病毒型特有的九个毒力基因，每个组都有代表（即EHEC的stx1和stx2，ETEC的lt、sth和stp，EPEC的eaeA和bfpA，EAEC的aggR，以及EIEC的ipaH）。PCR引物使用MultAlin构建。使用从代表每组的致泻性大肠杆菌菌株中分离的DNA来测量构建的多重PCR引物的灵敏度和特异性。检测限如下：EHEC为5×10¹ CFU/ml，表达lt和sth的ETEC为5×10³ CFU/ml，表达stp的ETEC为5×10⁴ CFU/ml，EPEC为5×10² CFU/ml，EAEC为5×10⁴ CFU/ml，EIEC为5×10² CFU/ml。为了确认特异性，空肠弯曲菌、产气荚膜梭菌、鼠伤寒沙门氏菌、副溶血性弧菌、单核细胞增生李斯特菌、小肠结肠炎耶尔森菌、蜡样芽孢杆菌和金黄色葡萄球菌用作阴性对照，未获得这些菌株的扩增产物。此外，该试剂盒使用来自腹泻患者的100份粪便样本和在韩国分离的150株致泻性大肠杆菌菌株进行了验证。总之，本研究中开发的多重PCR检测方法对于快速、特异性地检测五种致泻性大肠杆菌类型非常有用。这种一步检测方法将作为一种快速且经济的方法有用，因为它降低了鉴定致泻性大肠杆菌所需的成本和时间。
