食品中沙门菌特异性二重聚合酶链反应检测方法的研究

目的建立二重聚合酶链反应(PCR)方法快速检测食品中的沙门菌(Salmonella spp.)。方法以沙门菌特异性基因invA、hilA为靶基因,选择2对引物对16株沙门菌和24株非沙门菌进行扩增,验证二重PCR的特异性。梯度稀释DNA,以不同稀释度的DNA PCR扩增。在猪肉中以不同菌量人工污染,不同增菌时间培养,提取DNA进行PCR扩增。应用该方法检测实际样品。结果以invA、hilA为靶基因的两对引物对沙门菌的检出均有很好的特异性,PCR能检出0.1332pg的沙门菌DNA,人工污染猪肉的检测限为2.5cfu/ml。本研究共检测了53份食品样品,有21份检出了沙门菌。结论建立了适用于肉类、水产品及蛋糕等食品中的沙门菌检测的快速、灵敏、特异的二重PCR方法。

Study on a dulplex specific detection of Salmonella spp. in foods by a dulplex PCR

OBJECTIVE: To develop a rapid dulplex PCR assay for detection of Salmonella spp. in foods. METHODS: Two sets of primers that specific amplified segments of the invA, hilA genes were designed to detect Salmonella spp. Sixteen Salmonella spp. and twenty-four non Salmonella spp. were amplified by PCR to confirm the specificity. The genomic DNA was serially diluted and subjected by PCR amplification to verify sensitivity. Various numbers of bacteria were added into pork. After different time of enrichment, DNA was extracted and amplified by PCR to verify detection limit. This method was applied to detect naturally contaminated samples. RESULTS: The PCR system were specific. The detection limit of the sensitivity was 0.1332pg with genomic DNA and 2.5 cfu/ml in artificially contaminated pork. Fifty-three foods were detected by the dulplex PCR and twenty-one were found to be positive. CONCLUSION: A rapid, sensitive and specific dulplex PCR assay could be used for detection of Salmonella spp. in foods such as meat, aquatic products and cake.