双重PCR法检测冷冻淡水水产品中单核细胞增生性李斯特菌的研究

目的建立一种快速有效地检测出口冷冻淡水水产品中单核细胞增生性李斯特菌的方法。方法以李斯特菌属特异性iap基因和种特异性hly基因为靶序列,建立双重PCR方法;比较和优化3种提取模板的方法;通过干扰实验、模拟污染实验等验证了所建方法的稳定性、特异性、敏感性;并与SN0184-93方法和mini-VIDAS方法进行比较。结果建立的方法稳定性强、特异性和敏感性好,检测限度为3cfu/25g样品,检测结果与SN0184-93方法和mini-VIDAS方法的一致性为100%;该方法可直接使用单核细胞增生性李斯特菌菌液作为模板进行扩增。结论建立的双重PCR方法可快速、敏感,特异地检测出口冷冻淡水水产品中单核细胞增生性李斯特菌。

Detection of Listeria monocytogenes in exported fresh water food products by a duplex PCR assay

For the establishment of a rapid and effective method to detect the presence of Listeria monocytogenes in exported fresh water food products,the duplex PCR assay was developed using the genus-specific iap gene and species-specific hly gene of listeria as the target sequences.Three methods of DNA extraction were compared and optimized.The stability,specificity and sensitivity of the assay were illustrated by means of interference experiment and the mimic contamination experiment,and compared with SN0184-93 method and mini-VIDAS technique.It was demonstrated that the method of assay established showed high stability and excellent specificity and sensitivity,and its detection limit was 3 cfu per 25 g of samples.The consistency of this method with SN0184-93 method and mini-VIDAS technique approached up to 100%.It is apparent that the duplex PCR assay established is proved to be a rapid and effective method that can be used to detect L monocytogenes in exported fresh water food products.