实时荧光PCR同时检测丙型副伤寒沙门菌和猪霍乱沙门菌方法的建立和应用

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。

The development and application of real-time PCR assay based on modified molecular beacon for the rapid detection of Salmonella paratyphi C and Salmonella choleraesuis

Objective To develop real-time PCR assay for the simultaneous detection of S. paratyphi C and S. choleraesuis. The method would be applied to the rapid diagnosis and subtype of Salmonella. Methods Based on the sequences published in GenBank, primers and modified molecular beacon probes were designed for the simultaneous detection of S. paratyphi C and S. choleraesuis using real-time PCR. Results The real-time PCR assay based on modified molecular beacons had a high sensitivity. The limits of detection of pure DNA and culture were 10 fg / reaction and 20 CFU / reaction, respectively. There was no cross-reaction with other bacteria used as control. The assay was tested against 71 strains. No false signals were observed and the specificity was 100%. The blind test of 20 samples by the assay showed that 5 samples were Salmonella paratyphi C and 4 samples were Salmonella choleraesuis,the result was consistent with the tested samples. There was no Salmonella paratyphi C or Salmonella choleraesuis sample identified by the assay in 70 food samples, which was in accordance with the traditional detection. Conclusion The real-time PCR assay was rapid, sensitive and specific. It could be applied to the rapid diagnosis of S. paratyphi C and S. choleraesuis.