生鲜肉中3种食源性致病菌Taqman多重荧光定量PCR检测方法的建立

Establishment of a multiplex Taqman real-time PCR assay for the simultaneous detection of three foodborne pathogens in fresh meat

为了建立一种可快速特异检测生鲜肉中沙门氏菌、金黄色葡萄球菌、大肠杆菌O157:H7的多重实时荧光定量PCR方法,试验针对沙门氏菌invA基因、金黄色葡萄球菌nuc基因和大肠杆菌O157:H7 wzx基因的保守序列分别设计引物和Taqman探针,建立多重qPCR反应体系,进行特异性、灵敏度和重复性研究,用该方法检测30份生鲜肉中的3种食源性致病菌,并与国标法进行比较。结果表明:该方法只扩增3种靶细菌,对其它供试菌不扩增;金黄色葡萄球菌和沙门氏菌的检测灵敏度均为105拷贝数/μL,大肠杆菌O157:H7的检测灵敏度为104拷贝数/μL;该方法的重复性良好;对30份生鲜肉进行检测,检出3份沙门氏菌、4份金黄色葡萄球菌和7份大肠杆菌O157:H7,与国标检测方法相比,大肠杆菌O157:H7和金黄色葡萄球菌各有1份样品不符合,其它阳性样品完全一致。说明建立的基于Taqman探针的多重荧光定量PCR检测方法可以特异、快速地实现对生鲜肉中沙门氏菌、金黄色葡萄球菌、大肠杆菌O157:H7的检测。

In order to develop a rapid and specific multiplex real-time PCR assay for detection of Salmonella,Staphylococcus aureus and Escherichia coli O157:H7 in fresh meat.The primers and Taqman probes were designed with invA,nuc and wzx gene as target sequence,corresponding Salmonella,Staphylococcus aureus and Escherichia coli O157:H7 for the multiplex real-time PCR assay.The specificity,sensitivity and reproducibility of the assay were evaluated.Thirty fresh meat samples were screened for the 3 foodborne pathogens applying the assay compared with national standard.The results showed that the multiplex real-time PCR assay was able to detect the 3 target pathogenic bacteria,whereas other bacteria were not recognized.The sensitivity of the assay for Staphylococcus aureus,Salmonella and Escherichia coli O157:H7 was 105 copies/μL,105 copies/μL and 104 copies/μL,respectively.The repeatability of this assay was good.Among the 30 samples,3 positive for Salmonella,4 positive for Staphylococcus aureus,7 positive for Escherichia coli O157:H7.All positive samples were consistent with the national standard method except for one Escherichia coli O157:H7 positive sample and one Staphylococcus aureus positive sample.The results suggested that the Taqman probes-based multiplex real-time PCR assay is specific and rapid for detection of Salmonella,Staphylococcus aureus and Escherichia coli O157:H7 in fresh meat.