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目的建立伤寒和甲、乙、丙型副伤寒沙门菌的快速和特异的检测方法,用于沙门菌属内的分型鉴定。方法根据GenBank公布的伤寒和甲、乙、丙型副伤寒沙门菌的保守序列设计引物和改良分子信标探针,建立多重PCR检测方法和实时荧光PCR检测方法。结果采用所建立的多重PCR方法可分别检测到伤寒的3条特异性条带、甲型副伤寒沙门菌的2条特异性条带和丙型副伤寒沙门菌的1条特异性条带,但是乙型副伤寒沙门菌未出现特异性条带。建立的实时荧光PCR方法可以快速、特异、灵敏地检测出伤寒和甲、乙、丙型副伤寒沙门菌,纯DNA和菌液的最低检出限分别可达10 fg/reaction和20 CFU/reaction;对77株细菌的检测正确率达100%。结论建立的实时荧光PCR检测方法比多重PCR方法更能快速、特异、灵敏地检测伤寒和甲、乙、丙型副伤寒沙门菌。
Objective To establish a rapid and specific method for the detection of typhoid and Salmonella typhimurium A, B and C for the identification of Salmonella genotypes. Methods According to the published sequences of typhoid fever and Salmonella typhi isolated from GenBank, primers and modified molecular beacon probes were designed and used to establish multiplex PCR and real-time PCR. Results Three specific bands of typhoid fever, two specific bands of Salmonella paratyphi A and one specific band of Salmonella paratyphi A were detected by the multiplex PCR method. However, Salmonella paratyphi B does not appear specific bands. The established real-time fluorescence PCR method can detect typhoid and Salmonella paratyphi A, B, and C rapidly, specifically and sensitively. The minimum detection limits of pure DNA and bacterial liquid can reach 10 fg / reaction and 20 CFU / reaction respectively The detection rate of 77 strains of bacteria was 100%. Conclusion The established real-time fluorescence PCR detection method can detect typhoid fever and Salmonella paratyphi A, B and C more rapidly, more specifically and more sensitively than the multiplex PCR method.