低温肉制品中3种食源性致病菌多重聚合酶链式反应快速检测方法的建立  

Multiplex Polymerase Chain Reaction Assay for Rapid Detection of Three Foodborne Pathogens in Low-Temperature Meat Products

作　　者：李新福[1,2] 王周平[1] 李聪[1,2] 方伶俐[1] 赵颖 徐宝才[1,2,3] 

机构地区：[1]江南大学食品学院,江苏无锡214122 [2]江苏雨润肉食品有限公司,肉品加工与质量控制国家重点实验室,江苏南京211806 [3]江苏省肉类生产与加工质量安全控制协同创新中心,江苏南京210095

出　　处：《肉类研究》2017年第2期45-50,共6页Meat Research

基　　金：国家自然科学基金面上项目(31571909);“十三五”国家重点研发计划重点项目(2016YFD0400703-05)

摘　　要：针对低温肉制品中较易污染的沙门氏菌(Salmonella spp.)、单增李斯特菌(Listeria monocytogenes)和金黄色葡萄球菌(Staphylococcus aureus)等有害微生物,利用多重聚合酶链式反应(multiplex polymerase chain reaction,m-PCR)的方法,对3种菌同时快速检测,从而达到节省时间,提高效率的目的。使用沙门氏菌的inv A基因、金黄色葡萄球菌的Prf A基因和单增李斯特菌nuc基因设计3对特异性引物,在确定特异性引物的基础上,将3种菌进行培养增菌后接种于低温肉制品中,过夜富集后收集并进行灵敏度检测,优化多重PCR反应体系并应用于实际样品的检测。结果表明:多重PCR方法在同时检测这3种有害微生物时,m-PCR最佳反应条件如下:25 μL反应体系、10×PCR缓冲液2.5 μL、2.5 mmol/L d NTPs 2.0 μL、2.5 U/μL的DNA聚合酶0.5 μL及上、下游引物各0.5 μL、单增李斯特菌和金黄色葡萄球菌的模板1.0 μL、沙门氏菌的模板2.0 μL,加dd H_2O补足至25 μL,最佳退火温度为64℃。多重PCR快速检测方法适用于低温肉制品中沙门氏菌等3种食源性致病菌的检测,具有快速、灵敏、特异、简便等特点。Salmonella spp., Listeria monocytogenes （L. monocytogenes） and Staphylococcus aureus （S. aureus） are 3 species of bacteria which can easily contaminate low-temperature meat products. The objective of this study was to develop a rapid and efficient method for the simultaneous detection of the bacterial pathogens using multiplex polymerase chain reaction （m-PCR）. The specific primers for the invA gene of Salmonella spp., the PrfA gene ofL. monocytogenes and the nuc gene of S. aureus were designed and their specificity was determined. After enrichment culture, the 3 strains were inoculated to low-temperature meat products and incubated overnight for evaluating the sensitivity of the detection method. Optimization of the PCR system and its application to the detection of real samples were carried out. Our experimental results showed that the optimum PCR conditions were determined as follows： using a reaction system containing 2.5μL of 10 x PCR buffer, 2.0μL of 2.5 mmol/L dNTPs, 0.5μL of 2.5 U/μL DNA polymerase, 0.5μL of upstream primer, 0.5μL of downstream primer, 1.0μL of L. monocytogenes template, 1.0 μL of S. aureus template or 2.0μL of Salmonella spp. template brought with ddH2O up to 25μL, and an annealing temperature of 64℃. The multiplex PCR method was applicable to detect Salmonella spp. in meat products with the advantages of rapidity, high sensitivity and specificity and simplicity.

关 键 词：低温肉制品 多重PCR 快速检测 食源性致病菌 

分 类 号：TS251.7[轻工技术与工程—农产品加工及贮藏工程]