山羊4种腹泻病原菌多重PCR检测方法的建立

Establishment of multiplex PCR for detection of four kinds of diarrhea pathogenic bacteria in goats

为建立检测引起山羊腹泻的4种主要病原菌的多重PCR方法,本研究针对产肠毒素大肠杆菌(ETEC)K99基因,沙门菌invA基因,志贺氏菌侵袭性质粒H(ipaH)基因和奇异变形杆菌ureR基因设计4对特异性引物,通过对反应条件优化,建立快速检测引起山羊腹泻的4种主要病原菌的多重PCR方法,并应用该方法检测山羊腹泻样品。结果显示该多重PCR方法对巴氏杆菌等其它6种细菌无扩增;其敏感性分别为ETEC103cfu/mL、沙门菌102 cfu/mL、奇异变形杆菌102 cfu/mL、志贺氏菌103 cfu/mL;经检测该方法稳定性也好。应用该多重PCR方法对21份山羊临床腹泻样品的检测结果与细菌分离鉴定结果一致,无漏检。本研究建立的多重PCR方法可实现从山羊粪便样品中同时检测4种腹泻病原菌,对山羊腹泻病原菌的快速检测提供了技术支持。

This study was to establish a multiplex-PCR method for detection of four pathogens causing goat diarrhea. In this study, four pairs of primers were designed based on the k99 gene in ETEC, invA gene in Salmonella spp, ipaH gene in Shigella spp and ureR gene in Proteus mirabilis. The concentrations of the primes and the anneling temperatures were optimized to determine the reaction conditions, and the multiplex PCR was used to detect goat diarrhea samples. The results showed the multiplex PCR amplification bands were accordant with the expected bands, and those of 6 other pathogens were detected negatively. Under the optimized conditions, the detection sensitivity of the multiplex PCR for the four pathogens was 103 cfu/mL for Enterotixigenic Escherichia coli, 102 cfu/mL for Salmonella spp, 102 cfu/mL for Proteus mirabilis and 103 cfu/mL for shigella spp, and the stability of the method was good. The multiplex PCR method was applied to detect 21 goat diarrhea samples, and the results were consistent with those of bacteria isolation and identifiction. In this study, the multiplex PCR method can detect four kinds of diarrheal pathogens from goat feces, which provides technical support for rapid detection of goat diarrhea pathogens.