毒力基因表达差异的致泻大肠埃希氏菌聚合酶链反应检测方法的优化

Optimization of polymerase chain reaction detection method for diarrheagenic Escherichia coli with differences in virulence gene expression

中文关键词:  致泻大肠埃希氏菌  毒力基因  PCR扩增  凝胶电泳

英文关键词:diarrheagenic Escherichia coli  virulence gene  polymerase chain reaction amplification  gel electrophoresis

中文摘要:

      目的 优化GB 4789.6—2016《食品安全国家标准 食品微生物学检验 致泻大肠埃希氏菌检验》中的致泻大肠埃希氏菌聚合酶链反应(polymerase chain reaction, PCR)检测方法。方法 选取9株致泻大肠埃希氏菌标准菌株, 针对13种毒力基因和1个管家基因, 采用PCR扩增结合琼脂糖凝胶电泳进行检测分析; 电泳染色采用GelRed泡染法。结果 GB 4789.6—2016中提示为等效基因的escV/eae和ipaH/invE毒力基因在部分标准菌株中存在表达差异; 采用GelRed泡染法的琼脂糖凝胶电泳可获得更清晰、特异性更强的电泳图谱。结论 建议在使用国家标准方法鉴定致泻大肠埃希氏菌型别时, 应同时靶向escV和eae基因、ipaH和invE基因, 并采用GelRed泡染技术, 以提高检测结果的准确性和可靠性。

英文摘要:

      Objective To optimize the polymerase chain reaction (PCR) detection method for diarrheogenic Escherichia coli in GB 4789.6—2016 National food safety standard-Food microbiological examination-Examination of diarrheagenic Escherichia coli. Methods The 9 standard strains of diarrheogenic Escherichia coli were selected, and 13 kinds of virulence genes and 1 housekeeper gene were detected by PCR amplification and agarose gel electrophoresis; GelRed bubble staining was used for electrophoretic staining. Results The virulence genes of escV/eae and ipaH/invE, which were indicated as equivalent genes in GB 4789.6—2016, showed expression differences in some standard strains; a clearer and more specific electrophoretic map was obtained by agarose gel electrophoresis with GelRed staining. Conclusion It is recommended that when using the national standard method to identify the types of diarrheagenic Escherichia coli, both escV and eae genes, as well as ipaH and invE genes, shall be targeted simultaneously, and GelRed bubble staining technology shall be employed to enhance the accuracy and reliability of the detection results.