模拟粪便标本单增李斯特菌和伊氏李斯特菌TaqMan双重实时荧光定量-聚合酶链反应检测方法

目的建立一种TaqMan双重实时荧光定量-聚合酶链反应(real-time PCR),用于模拟粪便标本中单增李斯特菌和伊氏李斯特菌的快速检测。方法以单增李斯特菌特异基因hly和伊氏李斯特菌特异基因smcL作为靶基因,合成2对引物和其相应的荧光探针,制作标准曲线,建立从模拟粪便标本中直接检测单增李斯特菌和伊氏李斯特菌的TaqMan双重real-time PCR。利用李斯特菌属中其他种李斯特菌及常见致病菌验证引物、探针的特异性;通过制备模拟粪便标本并对其进行二次增菌后,分别提取DNA,采用TaqMan双重real-time PCR进行检测,以达到快速检测单增李斯特菌和伊氏李斯特菌的目的。结果采用本研究建立的TaqMan双重real-time PCR对模拟粪便标本的检测结果显示特异性良好,与其他种李斯特菌和其他病原菌均无交叉反应。模拟粪便标本中单增李斯特菌和伊氏李斯特菌的检测下限分别为2.45×103cfu/g和2.92×103cfu/g;模拟粪便标本在3 h内可得出检测结果,当模拟粪便标本中单增李斯特菌含量为6 cfu/g和伊氏李斯特菌含量为5 cfu/g时,经过增菌后使用双重real-time PCR可检测出阳性结果。结论本研究建立了以单增李斯特菌的特异基因hly和伊氏李斯特菌的特异基因smcL为靶基因的TaqMan双重realtime PCR检测方法,该方法具有特异性好、敏感性高、快速易操作等优点,可用于临床、食品及环境标本的快速诊断,以及我国人群中单增李斯特菌和伊氏李斯特菌的携带或感染状况的调查分析。

Novel multiplex real-time TaqMan PCR assay for detection of Listeria monocytogenes and Listeria ivanovii in simulated fecal samples

Objective To establish a novel multiplex real-time fluorescent TaqMan PCR assay for the rapid detection of Listeria monocytogenes and Listeria ivanovii in simulated feces samples. Methods One pair of primers and TaqMan probes were respectively designed on hly gene in L. monocytogenes and smcL gene in L.ivanovii. Standard curve was produced. A real-time PCR assay to detect fecal samples containing L. monocytogenes and L. ivanovii was established. The specificity of the primers and probes was tested by using other Listeria spieces strains and other entero-pathogenic bacteria. With two enrichment steps, DNA from fecal samples contaminated artificially with different concentrations of L. monocytogenes and L. ivanovii were examined by using real-time PCR and traditional detection tests. Results Positive results were detected in the samples containing L. monocytogenes and L. ivanovii, but negative in the samples of other bacteria. The lowest detection limits of this assay were 2.45103 cfu/g for fecal sample with L.monocytogenes, and 2.92103 cfu/g for fecal sample with L. ivanovii. The whole process can be finished within 3 hours for fecal samples. Positive results were obtained in the samples with 6 cfu/g of L. monocytogenes and 5 cfu/g of L. ivanovii by using real-time PCR assay after sample enrichment. Conclusion The novel multiplex real-time TaqMan PCR for the detection of L. monocytogenes and L. ivanovii in simulated fecal samples showed high specificity and simplicity, and this simple and reliable assay can be used in the rapid detection of clinical samples to facilitate the survey of carriage or infection status of L. monocytogenes and L. ivanovii in population in China.