RT-PCR方法检测单核细胞增生李斯特活菌研究

Detection of viable Listeria monocytogenes with RT-PCR assay

目的：建立单核细胞增生李斯特活菌快速检测技术，解决PCR带来的假阳性问题。方法：采用以mRNA为基础的RT—PCR方法对单核细胞增生李斯特氏菌的hlyA基因进行检测，设计引物，扩增，并作了特异性和灵敏度比较。结果：该引物能较好地扩增单核细胞增生李斯特菌的特异性片断，而对其它的李斯特菌以及常见食源性致病菌无特异性扩增条带；检测的灵敏度纯菌可达到1×10^4cfu／ml，人工污染样品猪肉、虾肉和牛奶等培养12—16h，可达4．5cfu／ml（g）。结论：可以应用该方法对样品中单核细胞增生李斯特活菌进行检测，能较好地解决PCR检测所带来的假阳性问题。

Objectlve：To develop a method for rapid detection of viable Listeria monocytogenes. Methods：The hlyA gene of Listeria monocytogenes was detected by mRNA - based RT - PCR. The sensitivity and specificity of the RT - PCR method were compared to conventional methods. Results： Only special fragment of Listeria monocytogenes could be extended by the primers. There was no crossreaction with Listeria and other food - borne pathogenes. The sensitivity of detection was 4. 5 cfu/ml （g）. Conclusion：This method can be used to detect viable Listeria monocytogenes in the samples to solve paise positive problems.