土壤环境中肠道致病菌的多重PCR检测研究初探

Detection of pathogenic enterobacteria in soil by multiplex-PCR

建立同时检测大肠杆菌、沙门氏菌、金黄色葡萄球菌、福氏志贺氏菌、铜绿假单胞菌等5种土壤常见肠道致病菌的多重PCR检测技术,为这些肠道致病菌感染的快速诊断提供实验依据.根据这些肠道病原菌的毒素基因、高度保守基因及特异性基因分别合成5对特异性引物,应用PCR扩增技术对目的菌株进行特异性检测.实验结果表明,5对寡核苷酸引物都具有较高的特异性和专一性,多重PCR检测限达到104cfu·g-1.多重PCR应用于土壤样品分析,极大的缩短了检测时间(仅需3～4h)、降低了检测成本,对控制病原菌的传播具有重要意义,可推广应用于环境监测、水源检测、食品卫生监督、商品检验检疫等领域.

To provide an experimental evidence for rapid diagnosis of those pathogenic infection, multiplex-PCR assay was conducted to simultaneously detect Escherichia coli, Salmonella, Staphylococcus aureus, Shigella flexneri and Pseudomonas aeruginosa in soil. According to the toxin genes, the high-conservative genes and specific genes of the pathogenic enterobacterial strains, five primer sets were selected to simultaneously detect the pathogenic enterobacterial strains by multiplex-PCR method. The results showed that the multiplex-PCR using these five primer sets produced specific amplicons of expected sizes and the detection limits for the bacterial targets were estimated at 104cfu·g^-1. Multiplex-PCR analysis, applied in analysis of soil samples, could greatly shorten the detection time （only need 3 to 4 hours）. It provided a cost-effective way to detect and control the transmission of pathogens and also could be extended and applied to other fields, such as environmental inspection, water detection, food sanitation supervision, commodity inspection and detection, etc.