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目的建立丙型副伤寒沙门菌和猪霍乱沙门菌的实时荧光PCR快速检测方法,用于沙门菌属内的分型鉴定。方法根据GenBank公布的丙型副伤寒沙门菌和猪霍乱沙门菌的保守序列,设计引物和改良分子信标探针,建立实时PCR检测方法。结果检测体系灵敏度高,纯DNA和菌液的最低检出限分别可达10fg和20CFU/反应体系;特异性好,对71株细菌的检测符合率达100%。20株沙门菌采取盲号模拟血培养标本进行血培养检测及鉴定,检出5株丙型副伤寒沙门菌和4株猪霍乱沙门菌,与试验的菌株相符。70份食品中用实时荧光PCR同时检测丙型副伤寒沙门菌和猪霍乱沙门菌均为阴性,而用传统方法分离培养未检出。结论建立的实时PCR检测方法可以快速、特异、灵敏地检测出丙型副伤寒沙门菌和猪霍乱沙门菌。
Objective To establish a rapid real-time PCR method for the detection of Salmonella paratyphi C and Salmonella choleraesu in Salmonella. Methods According to the conserved sequences of Salmonella paratyphi A and Salmonella choleraesuis published in GenBank, primers and modified molecular beacon probes were designed and used to detect real-time PCR. Results The sensitivity of the detection system was high. The detection limits of pure DNA and bacterial liquid were 10fg and 20CFU / reaction respectively. The specificity was good. The detection rate of 71 strains was 100%. Twenty strains of Salmonella were detected by blind culture of blood culture samples. Five strains of Salmonella paratyphi A and four strains of Salmonella choleraesuis were detected, which was consistent with the tested strains. 70 foodstuffs by real-time fluorescence PCR simultaneous detection of Salmonella paratyphi Salmonella and choleraesuis Salmonella were negative, but the traditional method of isolation and culture were not detected. Conclusion The real-time PCR assay can detect Salmonella paratyphi A and Salmonella choleraesuis rapidly, specifically and sensitively.