多重PCR法鉴定伤寒沙门菌的研究

Developed Method of a Multiplex PCR Assay for the Identification of Salmonella Enterica Serovar Typhi

目的建立多重PCR方法鉴定伤寒沙门菌,为临床确诊及现场流行病学调查提供快速准确的实验室技术支持。方法根据沙门菌菌体抗原A/D1群基因、鞭毛抗原基因(fliC-Hd)及伤寒沙门菌Vi抗原基因片段(Vi)设计引物,建立多重PCR体系并进行反应条件优化。选择15株不同血清型沙门菌及18株非沙门菌菌株,对所建立体系的特异性进行检测,并将该体系应用于浙江省分离的50株实际样本的检测。结果建立并优化了伤寒沙门菌检测的多重PCR体系,优化后25μl PCR体系包括100μM dNTPs、2.5 U Taq DNA聚合酶、引物各0.2μM、模板5μl;PCR条件为94℃变性1 min,56℃退火1 min,72℃延伸1 min,共循环35次。该体系具有高特异性,可准确快速鉴定伤寒血清型沙门菌,同时也可区分A群及D群血清群的沙门菌,并能检测鞭毛抗原为Hd及毒力基因Vi阳性的沙门菌,对实际样本检测符合率达100.00%。结论多重PCR可准确鉴定伤寒沙门菌,能作为传统血清学分型的辅助方法用于伤寒沙门菌的鉴定。

Objective To develop a multiplex PCR assay for the detection of Salmonella enterica Serovar Typhi.Methods According to the gene sequences of somatic antigen for salmonella serogroups A/D1,fliC-Hd and Salmonella Vi capsular antigen（Vi）,four pairs primers were designed.The multiplex PCR was developed and optimized.To valid the assay,genomic DNA from 15 salmonella strains representing 15 serotypes and 18 non-salmonella strains was subjected to the PCR.The method was applied to the detection of 50 samples isolated in Zhejiang Province.Results A multiple PCR was established to detect Salmonella enterica Serovar Typhi.The optimized reaction mixture（25 μl total volume） contained 100 μM dNTP mix,0.2 μM of each primer,2.5 U Taq DNA polymerase and 5 μl template.The cycling parameters of the multiplex PCR consisted of denaturation at 94 ℃ for 1 min,followed by 35 cycles at 94 ℃ for 1 min,56 ℃ for 1 min,72 ℃ for 1 min.This assay can differentiate Salmonella serogroup A from Salmonella serogroup D.and it also can identify the salmonella with fliC-Hd and the salmonella with Vi capsular antigen.The coincidence rate of the actual sample detection is up to 100.00%.Conclusion The multiplex PCR assay is highly selective and sensitive in detecting Salmonella Typhi and can be used as a supplementary method to traditional serum agglutination test.