三种食源性致病菌多重荧光PCR检测方法的建立

摘要

In this paper,we designed the specific primers and probes targeting conventional genes from Salmonella,Listeria monocytogenes and Campylobacter jejuni.After optimization of reaction conditions,a multiplex real-time PCR was developed.Specificity of each simplex real-time PCR assay was validated by testing various bacteria including positive isolates and negative isolates.The sensitivity of the triplex PCR assay was determined by detecting serially diluted Purified DNA and DNA extracted from bacterial culture by boiling.The results indicated that it is effective,specific,sensitive technique which can be used as a effective tool for the rapid detection of three foodborne pathogens such as Salmonella,Listeria monocytogenes and Campylobacter jejuni.%根据沙门氏菌、空肠弯曲杆菌和单核增生李斯特氏菌的保守基因序列,设计特异性引物和以不同荧光素标记的探针。通过对荧光PCR反应体系和反应条件的优化筛选,建立了检测沙门氏菌、空肠弯曲杆菌和单核增生李斯特氏菌的三重荧光PCR检测方法。为了评价所建立的实时PCR检测体系的特异性,试验中选取了阳性菌株及干扰菌株进行特异性验证。同时对梯度稀释的纯化DNA和不同浓度引物探针进行检测以确定方法的灵敏度。结果表明,该方法有效、特异、敏感、稳定,对于动物产品中沙门氏菌、空肠弯曲杆菌和单核增生李斯特氏菌的快速检测具有重要应用价值。