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发明公开
CN105821123A 基于单增李斯特菌毒力基因的三重实时荧光定量PCR检测用引物、MGB探针及检测方法 审中-实审

基于单增李斯特菌毒力基因的三重实时荧光定量PCR检测用引物、MGB探针及检测方法
摘要：
本发明公开了一种基于单增李斯特菌毒力基因的三重实时荧光定量PCR检测用引物、MGB探针及检测方法。本发明针对单增李斯特菌hlyA、inlA和plcB基因科学设计特异性引物，其DNA序列如SEQ ID NO.1～SEQ ID NO.6所示；同时提供MGB探针，其DNA序列如SEQ ID NO.7～SEQ ID NO.9所示。基于所述引物和MGB探针，本发明整体优化了实时荧光PCR检测体系和的反应程序条件，成功建立了高通量的单增李斯特菌的TaqMan ?MGB探针三重实时荧光定量PCR检测方法，操作简单、安全，最低检测细菌浓度为100CFU/mL，整个扩增过程在50min以内，具有更加高的灵敏度、特异性和稳定性，可以很好地满足食品安全中监管中单增李斯特菌的快速检测和鉴定，能快速、准确测定样本中单增李斯特菌的含量，同时可以分析单增李斯特菌菌株的三个毒力基因携带情况，可用于其流行病学溯源和毒力强弱分析。
摘要（英）：

The invention discloses a listeria monocytogenes virulence gene based primer, MGB (minor groove binder) probe and detection method for triple real-time fluorescent quantitative PCR (polymerase chain reaction) detection. According to the invention, the specific primer is scientifically designed for listeria monocytogenes genes hlyA, inlA and plcB, and DNA (deoxyribonucleic acid) sequences of the specific primer are as shown in SEQ ID NO.1-SEQ ID NO.6; and meanwhile, the MGB probe is provided and DNA sequences of the MGB probe are as shown in SEQ ID NO.7-SEQ ID NO.9. Based on the primer and the MGB probe, a real-time fluorescent PCR detection system and a reaction program condition are wholly optimized; a high-throughput listeria monocytogenes TaqMan-MGB probe triple real-time fluorescent quantitative PCR detection method is successfully established; the operation is simple and safe; the minimum bacterial detection concentration is 100 CFU/mL; the time for the whole amplification process is within 50 minutes; and the method has higher sensitivity, specificity and stability, can well meet quick detection and identification requirements of listeria monocytogenes in food safety supervision, quickly and accurately measure the content of the listeria monocytogenes in a sample and analyze the carrying situation of three virulence genes of a listeria monocytogenes strain, and can be used for epidemiological source-tracking and virulence analysis.

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