荧光实时定量PCR检测单核李斯特菌方法学建立及应用

目的 建立检测单核细胞增多性李斯特菌的荧光定量PCR方法，并利用此方法检测人工污染的牛奶中单增李斯特菌菌量。方法 选择单增李斯特菌特异性的基因Listeriolysin O基因为靶目标设计引物，采用煮沸法抽提纯单增李斯特菌株（1／2a）DNA，建立SYBRGREEN实时定量PCR检测体系：结果 定量PCR对李斯特菌可产生特异扩增信号，对其他菌（大肠杆菌）无响应。标准曲线的相关系数为0．9521。最小检菌量约为100个菌落形成单位／反应体系。在人为污染牛奶检测中，与传统细菌计数方法结果相比，相关系数为0．839。结论 实时定量PCR是一种快速、灵敏的定量检测单增李斯特菌的方法，可用于牛奶样品的检测。

Development and Application of Fluorescence Real - time Quantitative PCR for Detecting Listeria Monocytogenes

Objective Establishment of the fluorescence real-time quantitative PCR assay to detect Listeria monocytogenes(Lm) and used in quantification of bacterial burden in contaminated milk. Methods Primers were designed based on the Listeriolysin O gene which is specific for L.monocytogenes. Total DNA was isolated from pure L.monocytogenes strain by boiling method, and performed as template to optimize the condition of real-time PCR. A standard curve for quantification was plotted. Sterile milk was inoculated with L.monocytogenes, enriched and detected by quantitative PCR and conventional microbiology methods. Results L.monocytogenes were amplified by quantitative PCR and other bacteria (E.Coli.) (didn't) generate any signal. The correlation coefficient was 0.9521. The minimal detection limit was 100 cfu/reaction. The correlative coefficient was 0.839 between the PCR and conventional microbiology method applied in quantifying the experimentally contaminated milk. Conclusion Real-time quantitative PCR is a rapid and sensitive method which is capable for quantifying the L. monocytogenes in the milk.