沙门菌、产单核细胞李斯特菌多重PCR检测方法的建立及应用

目的建立沙门菌、产单核细胞李斯特菌多重PCR检测方法,提高检测效率和敏感性。方法选择沙门菌组氨酸转运操纵子基因、产单核细胞李斯特菌溶血素基因序列设计引物,在单一PCR方法基础上,建立检测两种病原菌的多重PCR技术。结果在495bp和850bp处分别出现预期的特异性DNA扩增条带,敏感性试验显示该体系能检测出32pg的沙门菌DNA和274pg的李斯特菌DNA,样品检测表明该方法与单一PCR和国标方法的符合率达100%。结论本研究建立了能同时检测沙门菌和产单核细胞李斯特菌的多重PCR技术,为主要食源性病原菌快速检测提供了新方法。

Establishment and application of multiple PCR technique for detection of Salmonella spp. and Listeria monocytogenes

To establish a multiple PCR technique for detection of Salmonella spp. and Listeria monocytogenes as to improve the efficiency and sensitivity of differential diagnosis for these infections, the specific primers for Salmonella were designed from gene sequence of histine trans-operon, and those for L. monocytogenes from hly gene, and a mutple PCR assay for detection of these two pathogens was established on the basis of simple PCR technique. It was found that the predicted specific DNA amplication bands could be demonstrated at sequences of 495 and 850 bps on 1.2% agarose gel. Assay on sensitivity of this multiple PCR technique revealed that it was able to detect as little as 32 pg DNA from Salmonella and 274 pg DNA from L. monocytogenes. The results of sample testing with multiple PCR, simple PCR as well as the international method for PCR were identical A procedure of simultaneous detection of two bacterial genes was thus established via multiple PCR. This method would be useful for the rapid detection of Salmonella and L. monocytogenes.