志贺菌、沙门菌和霍乱弧菌多重PCR快速检测体系的建立

目的 建立快速检测志贺菌、沙门菌和霍乱弧菌的多重PCR方法.方法 根据志贺菌ipaH基因、沙门菌ipaB基因及霍乱弧菌EPSM基因设计特异性PCR引物,加热煮沸法制备DNA模板,进行PCR扩增及琼脂糖电泳检测.结果 应用所建立的多重PCR方法能分别或同时快速、特异地检测出志贺菌606bp、沙门菌314bp和霍乱弧菌482bp的目的基因.结论 初步建立了灵敏、特异的一步法检测志贺菌、沙门菌和霍乱弧菌的多重PCR体系,可用于高危腹泻致病菌的早期快速诊断.

Establishment of a system for simultaneous detection of Shigella spp.,Salmonella spp. and Vibrio cholerae with multiplex PCR

Objective To establish a multiplex polymerase chain reaction (PCR) assay for simultaneous identification of Shigella spp., Salmonella spp. and Vibrio cholerae. Methods Based on the gene sequences of invasion plasmid antigen H (ipaH) in Shigella spp., invasion plasmid antigen B(ipaB)in Salmonella spp. and enterotoxin extracellular secretion protein (EPSM) in V. cholerae, three pairs of primer were designed. Genomic DNA was extracted by the boiling method and multiplex PCR was performed with premix Taq in an ABI 2720 thermal cycle. The PCR-amplified products were then analyzed by using agarose gel electrophoresis. Results Under the optimized conditions, the assay yielded a 606-bp product from Shigella spp., a 314-bp product from Salmonella spp., and a 482-bp product from V. cholerae, respectively. When the DNA extraction of multiple target organisms was included in the same reaction, two or three corresponding amplicons in different size were observed. Conclusions A rapid, specific and sensitive multiplex PCR system for simultaneous detection of Shigella spp., Salmonella spp. and V. cholerae has been established. The results suggest that the simultaneous amplification of several genes by multiplex PCR may provide an efficient and rapid diagnostic method for severe diarrhea.