重庆市4种高发肠道病原菌多重PCR检测方法的建立

Multiplex- PCR for detection of four frequent enteric pathogenic bacteria in Chongqing

目的建立一种能够同时快速检测样品中沙门菌、霍乱弧菌、金黄色葡萄球菌和副溶血性弧菌的多重PCR检测方法。方法根据霍乱弧菌ctx基因、副溶血性弧菌gyrB基因、沙门菌invA基因、金黄色葡萄球菌nuc基因设计引物,进行PCR扩增。同时对引物的浓度、Mg2+浓度、退火温度进行优化,并测定方法的特异性及灵敏度。结果该多重PCR体系能同时检测4种目的菌;灵敏度最低为100 cfu/ml;特异性较强。结论初步建立了能够同时快速检测样品中4种致病菌的多重PCR检测方法,适宜对重庆地区细菌性突发公共卫生事件的快速检测。

Objective To establish a multiplex PCR based system for rapid detection of Salmonella spp, Vibrio cholerae, Vibro parahaemolyticus and Staphylococcus capitis. Methods Four sets of primers were designed to amplify the gene segments of ctx of Vibrio cholerae, gyrB of Vibro parahaemolyticus, nuc of Staphylococcus capitis and invA of Salmonella spp. , then the concen tration of primers, Mg2 ＋ and annealing temperature were optimized to determine the specificity and sensitivity of the system. Re sults Four target bacteria were detected at the same time by multiplex PCR. The lowest sensitivity was 100 efu/ml and the specificity was high. Conclusion A rapid multiplex PCR based system has been successfully established for detection of four pathogenic bacteria, and the system is suitable for epidemic situation inChongqing.