生鲜猪肉中含扩增内标的沙门氏菌Real-time PCR检测方法的建立

摘要

The aim of this study was to develop a real-time PCR method including an internal amplification control (IAC) for rapid detection of Salmonella in fresh pork. The primers and probes were designed based on ttrBCA gene of Salmonella. IAC, which had the same primer as Salmonella DNA, was cloned from nucleocapsid protein of canine distemper virus. Annealing temperature and concentrations of primer and probe were optimized. The results indicated that all 19 non-Salmonella strains and 36 Salmonella strains could be correctly identified by the method. The detection limit was 100 CFU/ml by thermal lysis method or 2. 66 copies/μl using DNA extraction kit. Adding 103 copies of IAC in 25 μl reaction system did not affect the amplification of target gene. It took approximately only 12 h to detect the inoculated Salmonella strain in fresh pork by real-time PCR method.%建立含扩增内标的生鲜猪肉中沙门氏菌Real-time PCR检测方法,以提高检测的准确性.根据沙门氏菌ttrBCA基因设计引物和探针,利用犬瘟热病毒核衣壳蛋白基因构建和目标基因相同引物的扩增内标,并对退火温度和引物、探针的浓度进行优化,建立含扩增内标的Real-time PCR检测方法,用该方法对人工污染生鲜猪肉样品进行检测.结果显示,该方法对19株非沙门氏菌检测结果均为阴性,对36株沙门氏菌检测结果均为阳性,特异性较好,热学裂解制备DNA模板法检测灵敏度达100 CFU/ml,试剂盒提取DNA制备模板法检测灵敏度达1μl2.66拷贝.在25μl反应体系中加入扩增内标103拷贝不影响目标基因的扩增,该方法能在12 h内对人工污染沙门氏菌的肉样做出检测.该方法不仅可快速检测生鲜猪肉中的沙门氏菌,同时避免了假阳性现象的产生.