食品中致泻大肠埃希氏菌多重实时荧光PCR检测方法的建立与验证

Establishment and Validation of Multiplex Real-time Fluorescent PCR Method for Detection of Diarrheal Escherichia coli in Food

本研究建立了食品中致泻大肠埃希氏菌的多重实时荧光PCR定性检测方法,通过检测多种类人工污染样品进行方法验证。设计并筛选致泻大肠埃希氏菌14个目标基因的引物和探针,确定多重实时荧光PCR反应条件,同时用PCR和多重实时荧光PCR方法检测208株菌的14个目标基因,对不同浓度的阳性样本进行多重实时荧光PCR重复性检测,对结果进行灵敏度、特异性、准确度及重复性分析。PCR和多重实时荧光PCR方法检测7种食品类别的15种人工污染食品基质,验证多重实时荧光PCR方法在食品检测致泻大肠埃希氏菌的可行性。多重实时荧光PCR方法的灵敏度为100%,uidA特异性为97.1%,准确度为99.5%,eae特异性为94.8%,准确度为95.2%,其余12个目标基因的特异性、准确度均100%,两种检测方法的结果判定一致。多重实时荧光PCR方法在检测范围内各浓度水平上变异系数均小于2%,线性关系良好。结果表明,建立的多重实时荧光PCR方法能够灵敏、特异、准确、高效地定性检测食品中致泻大肠埃希氏菌。

A multiplex real time fluorescent PCR method for the qualitative detection of Diarrheal Escherichia coli(DEC)in food was developed in this study.The method was verified by detecting various kinds of artificially contaminated food samples.Primers and probes of 14 target genes of diarrheal Escherichia coli were designed and screened.The reaction conditions for multiplex real-time PCR were determined.Both PCR and multiplex real-time PCR methods were employed to detect 14 target genes of 208 bacterial strains,and the reproducibility of multiplex real-time PCR was tested on positive samples of different concentrations.Sensitivity,specificity,accuracy,and reproducibility analyses were performed on the results.The applicability of the multiplex real-time PCR method in detecting DEC in seven categories of food was validated.The sensitivity of the multiplex real-time PCR method was 100%,uidA specificity was 97.1%,with an accuracy of 99.5%.The eae specificity was 94.8%,with an accuracy of 95.2%.The specificity and accuracy for the remaining 12 target genes were both 100%.Results from both detection methods were consistent.The coefficient of variation for all concentration levels within the detection range of the multiplex real-time PCR method was less than 2%,indicating good linearity.The results indicate that the established multiplex real-time PCR method is sensitive,specific,accurate,and efficient for qualitative detection of DEC in foods.