沙门菌、巴氏杆菌和大肠埃希菌多重PCR检测方法的建立

Establishment of Multiplex PCR Detection Method for Salmonella,Pasteurella and Escherichia coli

旨在建立一种可以应用于临床样本同时检测沙门菌、多杀性巴氏杆菌和大肠埃希菌感染的多重PCR方法。根据NCBI上已收录的沙门菌hut基因、多杀性巴氏杆菌kmt1基因和大肠埃希菌23SrRNA基因的全基因序列,设计并合成3对特异性引物,通过优化多重PCR反应条件,建立能够同时检测3种细菌混合感染的多重PCR诊断方法。特异性分析表明,应用该方法可以从沙门菌、多杀性巴氏杆菌和大肠埃希菌以及3种细菌的混合物中扩增出3条大小分别为286bp、457bp和652bp的特异性条带,其他对照组检测结果均为阴性;敏感性分析表明,该方法对沙门菌、多杀性巴氏杆菌和大肠埃希菌的最低检出量分别为1.77×104、1.99×104、2.01×104 CFU/mL;人工模拟感染样本检测表明,该方法能从混合感染的病料中检测出3种病原菌。可见:所建立的多重PCR方法具有特异性强,敏感度高,稳定性好的特点,可以有效检测沙门菌、多杀性巴氏杆菌和大肠埃希菌的混合感染。

A multiplex PCR assay was developed and evaluated for the clinical detection of Salmonella spp,Pasteurella and Escherichia coli. Three pairs of primers have been designed and synthesized based on the highly conserved regions of the Salmonella spp hut gene, Pasteurella kmt 1 gene and Escherichia coli 23SrRNA gene. The multiplex PCR reaction condition was optimized and the multi plex PCR method for detecting the co-infection of Salmonella spp, Pasteurella and Escherichia coli was established. The multiplex PCR have been tested for specificity, sensitivity, repeatability and ar- tificial simulation tests. The specificity test showed that a fragment of 286 bp, 457 bp and 652 bp were amplified from Salmonella spp, Pasteurella and Escherichia coli, respectively. No amplification was achieved from control groups of other bacteria. The sensitivity test showed that the multiplex PCR could detect 1.77 × 104 CFU/mL for Salmonella spp, 1.99 × 104 CFU/mL for Pasteurella and 2.01 × 104 CFU/mL for Escherichia coli, respectively. The artificial simulation test showed that the multiplex PCR could detect three kinds of pathogens from co-infection disease material. The result in dicated the multiplex PCR method had advantages of specificity, sensitivity, repetitive, and it could effectively detect the co-infection of Salmonella spp, Pasteurella and Esckerichia coli.