肠出血性大肠杆菌O157∶H7和霍乱弧菌的多重PCR检测

DETECTION OF ENTEROHEMORRHAGIC ESCHERICHIA COLI O157∶H7 AND VIBRIO CHOLERAE BY MULTIPLEX PCR ASSAY

目的 建立同时检测肠出血性大肠杆菌 (O15 7∶H7)和霍乱弧菌的多重PCR方法 ,为霍乱和肠出血性大肠杆菌感染的快速诊断提供实验依据。方法 首先选择O15 7∶H7的O抗原(rfbE)、H鞭毛抗原 (fliC)基因以及霍乱弧菌外膜蛋白 (ompW )和肠毒素A亚单位 (ctxA)基因特异的 4对引物 ,分别或共同对O15 7∶H7和霍乱弧菌进行PCR扩增 ,并以琼脂糖凝胶电泳检测PCR产物。结果 所有O15 7∶H7菌株均在 4 97bp和 6 2 5bp处出现O15 7rfbE基因和H7fliC基因扩增产物 ;霍乱菌株均出现 5 6 0bp、30 2bp的ompW和ctxA基因扩增产物 ,O15 7∶H7和霍乱弧菌共同扩增可出现 4条单一条带。结论 选择 4对特异引物的多重PCR方法可简便、特异、快速、灵敏地对O15 7∶H7和霍乱弧菌进行同时检测。

Objective To develop a rapid 、specific and sensitive method of multiplex PCR assay to detect both enterohemorrhagic Escherichia coli (EHEC) O157∶H7 and Vibrio cholerae simultaneously,so as to provide experimental basis for rapid diagnosis of O157∶H7 infection and cholera. Methods Four pairs of primers were designed from O antigen, H flagellar antigen genes of O157∶H7 and an outer membrane protein(ompW) gene and toxin sub-unit A (ctxA) gene of Vibrio cholerae. O157∶H7 and Vibrio cholerae were detected by conventional PCR and multiplex PCR assay. Agarose gel electrophoresis was used to examine PCR products. Results O antigen and H flagellar antigen genes amplification generated PCR products at both 497bp and 625 bp in all EHEC O157∶H7 strains , simultaneous amplification of ompW and ctxA genes generated PCR products at both 560 bp and 302 bp in all Vibrio cholerae. Other non O157∶H7 and non Vibrio cholerae failed to yield any PCR products under comparable conditions.Conclusion This multiplex PCR method is a highly specific, rapid and sensitive method which can simultaneously distinguish O157∶H7 and Vibrio cholerae.