三种肠道致病菌多重PCR方法的建立与初步应用

Establishment and Primary Application of Multiplex PCR for the Detection Method of Three Food-Borne Pathogenic Bacteria

本研究根据沙门菌(Salmonella spp.)的fimA基因,奇异变形杆菌(Proteus mirabilis)的idsC基因,迟缓爱德华菌(Edwardsiella tarda)的fimA基因设计合成了三对特异性引物,建立一种同时检测三种食源性肠道致病菌的多重PCR方法,体系中检测到沙门氏菌的灵敏度为8 CFU;体系中检测到奇异变形杆菌的灵敏度为53 CFU;体系中检测到迟缓爱德华氏菌的灵敏度为72 CFU。对多重PCR的反应条件进行优化并组装成快速检测试剂盒。通过对采集的饲料、肉品和水产品等样品的检测,结果表明建立的多重PCR方法具有灵敏度高、特异性强、方便快捷等优点,与分离鉴定方法符合率100%,为研发快速检测以上三种食源性肠道致病菌的试剂盒提供了重要技术保障。

Three pairs of specific Primers were designed according to the gene fimA of Salmonella spp., the gene idsC of Proteus mirabilis and the gene fimA of Edwardsiella tarda. They are used to establish multiplex PCR method for diagnosing three species food-borne pathogenic bacteria synchronous. In the system, the detection sensitivity of Salmonella spp, Proteus mirabilis and Edwardsiella tarda was 8 CFU, 53 CFU and 72 CFU respectively. The reaction conditions on the multiplex PCR were optimized and assembled into a rapid detection kit. Through the detection of the samples, such as the feed, meat and aquatic product, the detection results showed that the multiplex PCR method has the advantages of high sensitivity, high specificity and convenience. The coincidence rate was 100 percent with isolation and identification method. It provides an important technical support for the development of rapid detection kit of the above three kinds of food-borne pathogenic bacteria.