应用多重PCR检测冻虾类海产品中3种致病菌.docVIP

多重PCR快速检测水产品中3种食源性致病菌方法的建立 摘要：沙门氏菌(Salmonella typli)侵袭性抗原保守基因invA、副溶血性弧菌(Vibrio parahaemolyticus)目的基因irgB和单核细胞增生李斯特菌(Listeria monocytogenes)调控蛋白基因prfA，设计了3对特异性引物，建立了快速检测水产品中 3种主要食源性致病菌的多重 PCR方法。结果显示，沙门氏菌、副溶血性弧菌和单核细胞增生李斯特菌的多重 PCR 扩增片段产物大小分别为213、 369和517 bp。反应条件优化后，3种目的菌在103CFU/mL均可同时扩增出较清晰条带，彼此之间无交叉反应，具有良好的特异性和灵敏度。该方法应用于3份人工污染水产样品的检测，并与国标方法对比验证，3种致病菌的整体符合率均达100%。该方法操作简单，检测周期短，灵敏度高、特异性强，关键：多重PCR；沙门氏菌；副溶血性弧菌；单核细胞增生李斯特菌Establishment of multiple-PCR for diagnosing three foodborne pathogenic bacteria in aquatic products Xia Hui-li Zheng Hui Research Institute of Quality Technical Supervision and Inspection, Taizhou, Zhejiang, 318000, China Abstract: A rapid multiplex- PCR method was established in order to detect three foodborne pathogenic bacteria including Salmonella typli，Vibrio parahaemolyticus and Listeria monocytogenes in aquatic products. Three pairs of oligonucleotide primers were designed for multiplex-PCR amplification according to gene coding invasion protein A of Salmonella typli， gene coding immune response of Vibrio parahaemolyticus and regulation histone gene of Listeria monocytogenes. gene of Shigella spp． The amplified fragment sizes of these three bacteria were 213 bp, 369 bp and 517bp, respectively．Under the optimized conditions, the specificity of the multiplex- PCR was high, and the minimum detection limit was 103 CFU/mL. The multiplex- PCR method was used to analyze three aquatic product samples compared with national standard methods, the coincidence rate of two methods reached 100%. The method developed in this study had high sensitivity and specificity, which could be applied for the rapid detection and molecular epidemiology survey of foodborne pathogenic bacteria in aquatic products． Keywords: Mmultiple-PCR, Salmonella typli, Vibrio parahaemolyticus, Listeria monocytogenes, Aquatic products 0 引言 单核细胞增生李斯特菌在河口、海水及沉积物、水产养殖区域，寄生在海洋生物体中[]，通过污染的动物源性食品感染人类食源性致病菌的检测列为重要的卫生检出指标之一。 产品中沙门氏菌、副溶血性弧菌和单核细胞增生李斯特菌1 材料与方法 1.1 材料 1.1.1 标准菌株 本试验选用的试验菌种由广东环凯