Taqman三重实时PCR快速检测原料乳中致泻性大肠埃希氏菌

Taqman triplex real-time PCR assays for rapid detection of diarrheagenic Escherichia coli in raw milk

建立可对原料乳中致泻性大肠埃希氏菌(包括肠致病性大肠埃希氏菌、肠侵袭性大肠埃希氏菌和肠产毒性大肠埃希氏菌)同时进行快速检测的Taqman三重实时PCR方法。以致病菌中常见的eae、ipaH和lt基因为目的片段,选择3种特异性引物和Taqman探针进行三重实时PCR扩增,研究反应的特异性、检测限和重复性,并用所建立的方法对38份原料乳进行检测。结果表明,扩增曲线形态良好,对原料乳中其他常见致病菌无交叉反应,对含致病菌为1 cfu.mL-1的乳样经2 h增菌处理后检测结果呈阳性,对原料乳样重复性检测的CV值均小于5%。完成全部检测过程只需大约6 h。该方法快速准确,可应用于原料乳中致泻性大肠埃希氏菌的快速检测和污染调查。

Arapid triplex real-time PCR based on Taqman technique for the simultaneous detection of diarrheagenic Escherichia coli（including enteropathogenic E.coli,enteroinvasive E.coli and enterotoxigenic E.coli） in raw milk was developed.Three kinds specific primers and Taqman probes were used to amplify the f requent target genes eae,ipaH and lt of pathogenic bacteria.Specificity,sensitivity as well as re-producibility of the Taqman triplex real-time PCR assays were studied,and diarrhea E.coli in 38 raw milk samples was detected by this method.The results showed taht amplification curve reflected well,and cross-reactivity was not seen with any of the negative control cultures of other food-borne pathogens found frequently in raw milk.When artificial polluted milk was cultivated for 2 h,in which concentration of pathogenic bacteria was 1 cfu.mL-1,detection result presented positive.Coefficient of variation of reproduci-bility assay for raw milk was less than 5%.The detection was finished in about 6 h.This method was rapid and reliable,and it was suggested that this method could be applied to diagnosis and detection ofenteropathogenic Escherichia coliin raw milk.