多重TaqMan qPCR快速检测致泻性大肠埃希氏菌方法的建立及应用

Establishment and application of a multiplex TaqMan qPCR method for rapid detection of diarrheagenic Escherichia coli

摘要:

为解决生化鉴定和现有PCR方法在对致泻性大肠埃希氏菌(Escherichia coli)进行检测及分型时所存在的操作繁琐、耗时长、存在假阳性等问题，本研究根据5类致泻性大肠埃希氏菌中包含的11个毒力基因设计引物及探针，建立了一种基于TaqMan法的多重实时荧光定量PCR检验方法，并对实验条件进行了优化，可对致泻性大肠埃希氏菌进行快速检测并进行分类。结果表明，本方法可从多种常见食源性致病性菌株中特异性检测出致泻性大肠埃希氏菌并进行分型，且检测所需DNA模板最低浓度仅为2.5 ng/μL；同时，通过人工污染试验证实，样品增菌培养时间不少于18 h时，即可实现有效检测。本检测方法具有高特异性、灵敏度、短时高效，适用于食品中致泻性大肠埃希氏菌的大批量快速检测。本方法可为食品安全领域提供新的技术支持，有助于进一步减少人力投入、提升检测效率。

Abstract:

To address the cumbersome operation, long time consumption, and false positive results of biochemical and PCR methods in the detection of diarrheagenic Escherichia coli (DEC), we designed primers and probes targeting 11 virulence genes harbored by five pathotypes of DEC. We then established a novel multiplex TaqMan qPCR method for the rapid detection and typing of DEC after optimization of the experimental conditions. The detection results demonstrated that this method could be used for specific identification and typing of DEC among various foodborne pathogenic strains. Furthermore, it can achieve effective detection when the DNA concentration is no less than 2.5 ng/μL or the culture duration is no less than 18 h. These findings indicate that the developed detection method boasts high specificity, high sensitivity, and short time consumption, which make it applicable for the industrial-scale rapid detection of DEC in food. This study provides novel technical support for food safety sector, which is conducive to further reducing labor input and improving detection efficiency.