乳制品中四种病原菌多重荧光PCR检测方法的建立与应用

Establishment and Application of Multiplex Fluorescent PCR for Detection of Four Pathogenic Bactria in Dairy Products

本研究建立了可快速特异检测乳制品中阪崎肠杆菌、沙门氏菌、金黄色葡萄球菌和单核细胞增生李斯特氏菌的多重荧光PCR方法。通过设计阪崎肠杆菌、沙门氏菌、金黄色葡萄球菌和单核细胞增生李斯特氏菌的特异性引物和探针,建立了多重PCR反应体系,评价了该方法的特异性、灵敏度和精密度,并用该方法和国标法分别检测了150份乳制品中的4种致病菌,对检测结果进行对比。结果显示,检测目标菌为相应阳性,检测其他12种非目标供试菌为阴性;检测灵敏度分别为1 cfu/μL;其精密度的变异系数均小于5%;与国标检测方法相比其检测结果具有良好的一致性。

In this study,a rapid,sensitive and specific multiplex fluorescent PCR method was developed to detect Enterobacter sakazakii,Salmonella enterica,Staphylococcus aureus and Listeria monocytogenes in dairy products.Through designing specific primers and probes of Enterobacter sakazakii,Salmonella enterica,Staphylococcus aureus and Listeria monocytogenes,the multiplex fluorescent PCR was established.The specificity,sensitivity and precision of this method were evaluated,while this method and national standard method were used to test 4species of pathogenic bacteria in 150 samples of dairy product,the results of which were compared.The results showed that those four targetbacteria were positive,meanwhile the other 12 non target bacteria were negative.The sensitivity of 4 species of pathogenic bacteria was detected as 1 cfu/μL.Precision test showed that coefficient variables were all less that 5%.Compared with national standard method,multiplex fluorescent PCR method had good consistency.