饮用水中5种致病菌多重PCR技术检测研究

Study on Detection of Five-kinds of Pathogenic Bacteria in Drinking Water by Multiplex-PCR

沙门氏菌(Salm onellasp.)、志贺氏菌(Shigellasp.)、绿脓杆菌(Pseudom onasaeruginosa)、肠出血型大肠杆菌O157(E terohaem orrhagicO157)和副溶血弧菌(Vibrio rah-aem olyticus)是5种饮用水中不得检出食源性致病菌,根据它们的毒素基因、高度保守基因及特异性基因,设计合成5对寡核苷酸引物,应用PCR技术对10个属的30株细菌进行引物特异性检测。通过对多重PCR反应体系、条件进行优化,显著提高了检测灵敏度。初步应用于水样分析中,极大的缩短了检测时间、降低了成本。实验结果表明:5对寡核苷酸引物都具较高的特异性和专一性,多重PCR检测灵敏度达到101~102cfu,检测需5~6h,在水样检测的初步应用中得到了均一、稳定、清晰的结果,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。

The five kinds of food-borne pathogenic bacteria including Salmonella sp.、Shigella sp.、Pseudomonas aeruginosa、Eterohaemorrhagic O157 and Vibrio rahaemolyticus that couldn't be detected in drinking water. The five pairs of primers were designed and composed according to the toxin genes, high-conservative genes and specific genes of these pathogens. The specificity of individual primer pairs in PCR was evaluated on DNA templates of more than thirty different bacterial isolates from ten species. Through optimizing the system and condition of multioplex-PCR, the detection sensitivity was improved dramatically. Multiplex-PCR analysis was primary performed on several water samples. The method established shortened the detection time and provided a cost-effective way in maximum. The experiment showed the multiplex-PCR using the five pairs of primers produced specific amplicons of expected sizes, the detection limits for the bacterial targets were estimated at 10^(1)~10^(2)cfu for one essay. It only took five to six hours to detect a sample, got the single, steady and clear result when the method was applied primarily in the analysis of water samples. The informative supplement to those areas which were environmental inspection, water detection, food sanitation supervision , commodity inspection and detection, etc.