Taq Man-MGB探针Real-timePCR快速检测单增李斯特菌的研究

目的：建立TaqMan—MGB探针Real—time荧光PCR快速检测单增李斯特菌技术。方法：在对单增李斯特菌invA序列进行分析、比较基础上，设计一对特异性TaqMan—MGB探针法引物及探针，通过Real—timePCR反应条件和反应体系的优化，实现对单增李斯特菌的快速检测；用克隆到pMD18-T载体上的李斯特菌invA基因阳参片段及不同菌株、食品标本验证方法的特异性和敏感性。结果：方法的灵敏性高，其循环阈值与模板浓度的对数值具有很好的对应关系，最低可检测57个拷贝数，经18h增菌，可检测低至4．5cfu／ml的细菌；特异性强，检测6株单增李斯特菌标准株和100株单增李斯特菌样品分离株的PCR循环域值（cT值）均小于25，而90株威氏李斯特菌、英诺克李斯特菌、绵羊李斯特菌以及非李斯特菌PCR循环域值（CT值）大于35；快速，最快1h可出结果，实际样品20h可出结果，而常规细菌培养法需要一周以上；对103份速冻米面食品进行检测，13份阳性，与常规方法无显著性差异（P〉0．05）。结论：TaqMan—MGB探针的单增李斯特菌Real—time荧光PCR检测方法具有特异性强，敏感性高，易操作等优点，可推广应用。

Application of Taq Man - MGB probe real - time fluorogenic PCR assay to detection of Listeria monocytogenes

Objective:To establish technique standard for the detection of Listeria monocytogenes by using Taq Man-MGB probe real-time fluorogenic PCR assay.Methods:A pair of specific Taq Man-MGB probe and primer was designed based on the invA sequence analysis and comparison of L.monocytogenes,and quick detection of L.monocytogenes was achieved by optimizing Real-time PCR reaction condition and system.The specificity and sensitivity of the present method were tested by invA gene segments cloned on pMD18-T vector,different strains of L.monocytogenes and food samples.Results:This method was proved to be sensitive and specific to detect L.monocytogenes efficiently.The Ct value and the template concentration have the good corresponding relations.It was demonstrated that the lowest detection limit of this method for bacterial suspensions of LM was 57 copies,while that for modified specimens was 4.5 cfu/ml after 18 h amplification.PCR CT value was lower than 25 when this method was used to detect 5 standard L.monocytogenes strains and 100 strains of L.monocytogenes isolated from samples.In contrast,PCR CT value was higher than 35 when used to detect other Listeria spp,including welshimeri,innocua,seeligeri and non-Listeria bacteria.The quickest was 1 h to get assay result while it took 20 h to get result if real samples were used,or over one week if conventional bacterial culture method was employed.The present method was tested in 103 samples of instant frozen flour food,13 samples were shown positive,and there was no significant difference as compared with routine method.Conclusion:The Taq Man-MGB probe real-time PCR technology for detecting L.monocytogenes shows high specificity,sensitivity and simplicity.Therefore,it is recommended for practical application.