针对单增李斯特菌iap基因PCR检测方法的建立及其应用

Establishment and application of the PCR method targeting the iap gene for the detection of Listeria monocytogenes

为了建立快速、简便的单增李斯特菌(Listeria monocytogenes,LM)PCR检测方法,试验根据Gen Bank上已发表的单增李斯特菌iap基因序列,设计1对特异性引物,通过优化各种条件建立针对iap基因的单增李斯特菌的PCR检测方法,并对该方法的灵敏度和特异性进行评估及初步临床应用。结果表明:建立的PCR检测方法的最佳退火温度为60℃,Mix最佳使用量为12.5μL,模板使用量为30 ng;该方法对单增李斯特菌纯培养物的检测限为1×105cfu/m L,且能够区分单增李斯特菌和英诺克李斯特菌,对沙门氏菌、大肠杆菌及猪链球菌均无交叉反应;对42份疑似感染单增李斯特菌临床样品进行PCR检测,有7份扩增结果呈阳性,与传统分离培养结果一致。说明试验所建立的PCR方法可用于食品中单增李斯特菌的检测。

To establish a fast and convenient PCR method for the detection of Listeria monocytogenes, a pair of specific primers was designed using the iap gene sequence published in GenBank. The PCR method targeting the iap gene for the detection of Listeria monocytogenes （ L. mono- cytogenes） was established through optimizing various conditions. Moreover, the sensitivity and specificity of the method were evaluated and pre- liminarily applied in the clinic. The results showed that based on the established PCR detection method, the optimal annealing temperature was 60 ~C, and the optimal volume of Mix was 12.5 μL, and the amount of DNA template was 30 ng. The detection limit of the method for L. monocytogenes pure culture was 1 × 105 cfu/mL, and the method could distinguish L. monocytogenes and Listeria innocua, which had no cross - reaction with Salmonella, Escherichia coli and Streptococcus suis. 7 from 42 suspected clinical samples were positive by the PCR method, which was accordance with the result of isolation and culture using traditional method. The results indicate that the PCR method established by the test can detect L. monocytogenes in food.