阪崎肠杆菌zpx基因的分子信标-实时PCR技术研究

A real-time PCR with molecular beacon assay for the detection of zpx gene of Enterobacter Sakazakii in food

目的建立分子信标-实时PCR技术检测婴幼儿乳粉中阪崎肠杆菌的快速方法。方法在PCR反应体系中加入分子信标探针,探针的5'端标记FAM,3'端标记TAMRA,建立阪崎肠杆菌zpx基因分子信标-实时PCR技术快速检测方法。结果检测方法特异性强,无非特异性扩增;分子信标-实时PCR反应体系DNA灵敏度为180fg/PCR反应体系,纯阪崎肠杆菌菌液的检出限为102 CFU/ml,无交叉反应;以此反应体系检测23份样品,其中2份为阳性,余未检出,与传统检测方法结果一致。结论分子信标-实时PCR检测体系快速、灵敏度高、特异性强,可用于婴幼儿乳粉中阪崎肠杆菌的快速检测。

Objective To develop a method for rapid detection of Enterobacter Sakazakii from contaminated food by real-Time PCR with molecular beacons.Methods The zpx gene of Enterobacter Sakazakii was amplified with PCR reaction system containing a molecular beacon probe labeled with FAM at its 5′end and with TAMRA at its 3′end;and then the reaction system was established for rapid detection of Enterobacter Sakazakii.Results The reproducibility of the method was good and no non-specific amplification.The sensitivity achieved 180 fg/PCR reaction system for the DNA and 103CFU/ml for the pure culture of Enterobacter Sakazakii..There was no cross-reaction with other bacteria,only 2 of 23 tested samples were positive,and the result of this assay was consistent with other traditional methods.Conclusion The real-time PCR with molecular beacon assay is rapid,sensitive,and specific for Enterobacter Sakazakii detection,and can be used for rapid microbiological analysis of Enterobacter Sakazakii in powdered infant formula.