TaqMan探针实时定量PCR检测肉中单增李斯特菌的研究

根据GenBank公布的单增李斯特菌的hlyA基因序列设计一对引物和一条TaqMan探针,在分析特异性基础上,构建阳性重组质粒进行荧光定量PCR扩增,制备标准曲线,分析敏感度。进一步将该方法应用到人工染菌肉样中,分析其最低检出限。结果表明,该方法对10株单增李斯特菌均可产生特异性扩增曲线,其他6株非单增李斯特菌不产生扩增曲线,表明该方法具有较强的特异性。对阳性重组质粒定量扩增所建立标准曲线的相关系数为0.998,敏感度为19.5 copies.μL-1。该法对人工染菌肉样中单增李斯特菌的最低检出限为2.8×102cfu.g-1。该方法特异性强、敏感性高,可为肉中单增李斯特菌的快速、准确的定量检测奠定基础。

Real-time TaqMan PCR for detection of Listeria monocytogenes in meat

The specificities of the primers and TaqMan probe that were designed according to the hlyA gene of Listeria monocytogenes were tested.The real-time PCR assay was developed by amplifying quatitatively hlyA gene and constructing standard curve of positive recombinant plasmids.Then the assay was applied in artificially inoculated meat.The results showed that all of the strains of Listeria monocytogenes(n=10) tested were positive and all other nonspecific strains(n=6) tested were negative.The correlation coefficient of the standard curve was 0.998 and the sensitivity was 19.5 copies.μL-1 in positive recombinant plasmids.Then the detection limit of 2.8× 102 cfu.g-1 was determined in artificially inoculated meat.The assay was specific and sensitive highly,which would be extended to detect quatitatively Listeria monocytogenes in meat.