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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 establish a multiplex PCR method for the identification of Salmonella typhi, providing rapid and accurate laboratory technical support for clinical diagnosis and field epidemiological investigation. Methods The primers of fliC-Hd and Vi of Salmonella typhimurium were designed according to the A / D1 group gene of Salmonella enterica and the multiplex PCR system was established and the reaction conditions were optimized. Fifteen strains of Salmonella serotypes and 18 strains of non-Salmonella were selected to test the specificity of the established system. The system was applied to the detection of 50 actual samples isolated in Zhejiang Province. Results The multiplex PCR system for the detection of Salmonella typhi was established and optimized. The optimized PCR system consisted of 100 μM dNTPs, 2.5 U Taq DNA polymerase, 0.2 μM primers and 5 μL template. The PCR conditions were denaturation at 94 ° C for 1 min, annealing at 56 ° C 1 min, 72 ℃ extended 1 min, a total of 35 cycles. The system has high specificity, rapid and accurate identification of Salmonella typhi serogroups, but also can distinguish between groups A and D serogroup Salmonella, and can detect flagella antigen Hd virulence gene Vi positive Salmonella, The actual sample detection rate of 100.00%. Conclusion Multiplex PCR can accurately identify Salmonella typhi, and can be used as an aid in the traditional serological typing in the identification of Salmonella typhi.