奶液模拟标本中单增李斯特菌real-time PCR检测方法的建立

Real-time PCR assay for rapid detection of Listeria monocytogenes in simulated milk specimens

目的建立了基于TaqMan探针的real-time PCR技术针对奶液模拟标本中单增李斯特菌的快速检测方法。方法用单增李斯特菌hlyO基因的部分片段作为靶基因,制作标准曲线,定量检测奶液中的单增李斯特菌。结果通过对不同李斯特菌及一些较为常见的致病菌的DNA进行扩增,只有单增李斯特菌能够产生扩增曲线,其余菌株均不产生扩增曲线。单增李斯特菌的检测灵敏度可以达到9copies/反应体系。结论该方法特异性好,灵敏度高,整个实验可在1.5h内完成,可用于食品中单增李斯特菌的快速检测和疫情暴发时的相关病原调查。

Objective To establish a real-time PCR assay for the rapid detection of Listeria monocytogenes in simulated milk specimens.Methods Based on part fragments of hlyO gene,a pair of primers and Taq-Man probe were designed for quantitative detection of L.monocytogenes.The specificity of the primers and probe were tested by using different L.monocytogenes strains and other common pathogenic bacteria.Results L.monocytogenes strains were positive in the detection and other tested strains were negative.The sensitivity of assay was 9 copies per PCR reaction.Conclusion The specificity and sensitivity of Taq Man real-time PCR technology for detecting L.monocytogenes in simulated dairy specimens were high,and the assay could be completed within 1.5 h.This method could be used to detect other food samples contaminated by L.monocytogenes and identify the cause of food-borne Listeriosis outbreaks.