单核细胞增生李斯特氏菌快速检测方法建立

ESTABLISHMENT OF RAPID DETECTION OF LISTERIAMONOCYTOGENES BY PCR

[目的]建立单核细胞增生性李斯特氏菌(Lm)快速检测(PCR)方法。[方法]用PCR特异性检测方法,根据Lm的inlA、plcA、plcB、hlyA基因设计4对相应的引物,检测不同浓度Lm纯培养物、金黄色葡萄球菌和乙型副伤寒沙门氏菌标准菌株的纯培养物以及Lm模拟污染的牛奶样品。[结果]Lm扩增出4个相应的产物(分别为255bp、129bp、260bp、234bp),金黄色葡萄球菌和乙型副伤寒沙门氏菌菌株均未扩增出特异性的片段,其中inlA基因最低检出限为25.5μg/ml,其他3对引物最低检测限达2.55μg/ml。对模拟污染的牛奶TSBYE增菌培养液用PCR检测,只扩增出3个相应基因的产物(plcA、plcB、hlyA)。[结论]扩增plcA、plcB、hlyA基因的PCR法对Lm的鉴定具有特异性强、灵敏度高、速度快和易操作等优点,可用于食品的Lm的分离。

[Objective] To establish a rapid PCR method for detecting isteriamonocytogenes. [Methods] By specificity detection method of PCR, four pairs of related primers were designed according to the inlA, pleA, plcB and hlyA gene of Lm to detect the different concentrations of pure culture with Listeriamonoeytogenes, pure culture with Staphylococcus aureus and Salmonella paratyphi B, and milk contaminated with Listeriamonocytogenes. [ Results] Four related production were amplified with Lm,they were 255 bp, 129bp, 260bp and 234bp, respectively. No specific fragment were amplified from Staphylococcus aureus and Salmonella paratyphi B. The limit of detection for inlA gene was 25.5μg/ml, the lowest detectable limit for other three pairs of primers was 2.55μg/ml. The enrichment culture solution of contaminated milk was detected by PCR, only three production of related genes were amplified including picA, plcB and hlyA. [Conclusion] PCR method which amplified picA, plcB and hlyA genes have the aspects of high specificity, high sensitivity, rapid and convenience, and cotdd be used to separate the Lm in food products.