实时RT-PCR检测存活于乳中的单核细胞增多性李斯特菌  

Detection of Viable Listeria monocytogenes in Milk by Real Time RT-PCR

作　　者：闫冰[1] 姜毓君[1] 曲妍妍[1] 毕宇涵[1] 相丽[1] 赵凤[1] 

机构地区：[1]乳品科学教育部重点实验室,东北农业大学食品学院,黑龙江哈尔滨150030

出　　处：《食品科学》2008年第2期292-296,共5页Food Science

基　　金：中国博士后科学基金项目(20060400803);黑龙江省科技攻关重大项目(GA06C101-08);黑龙江省高校青年学术骨干支持计划项目(1151G006)

摘　　要：单增李斯特菌是一种可在低温下生长、能引起人畜共患病的食源性致病菌。为克服传统PCR检测的假阳性问题,有效的检测单增李斯特活菌,本研究提取单增李斯特菌的总RNA并进行反转录,以单增李斯特菌的必要毒力基因hlyA为靶基因,设计特异性引物和TaqMan探针进行实时PCR检测,研究了检测的特异性和灵敏度,考查了对人工污染牛乳进行检测的应用性。结果表明,所用引物和探针能较好的扩增目的基因,而对其他食源性致病菌无交叉反应。单增李斯特菌经1h增菌,可检出3×102CFU/ml。而经3h增菌,活菌检测限可达30CFU/ml。人工污染牛乳样品经6h增菌,检测限为17CFU/ml。此方法可应用于乳中单增李斯特菌的快速检测和污染状况调查。Listeria monocytogenes is a kind of food-borne pathogenic bacterium strain grown at low temperature and can cause zoonosis. The method is based on real time reverse-transcripfion-PCR （RT-PCR） amplification of mRNA to detect viable Listeria monocytogenes by specific h/yA gene. This method can overcome the false-positive caused by amplification of nonviable Listeria monocytogenes in the detection process. Both sensitivity and specification of this method were studied, and the artificially contaminated milk by Listeria monocytogenes was assayed. The results showed that the specific primers and probe do not cross- react with other food-borne pathogens. The sensitivity of detection Listeria monocytogenes is 3 × 10^2 CFU/ml after being cultivated I h and 30 CFU/ml after 3 h. The sensitivity of detection of artificially contaminated milk is 17 CFU/ml after being cultivated 6 h. So this method can satisfy the demandion of diagnosis and detection of Listeria monocytogenes.

关 键 词：实时RT-PCR 单增李斯特菌 hlyA 人工污染乳 活菌 

分 类 号：TS207.4[轻工技术与工程—食品科学]