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目的:研究建立TaqM an双重荧光PCR技术,用于快速检测模拟临床标本中产毒型O139群霍乱弧菌。方法:以O139群霍乱弧菌O抗原编码基因rfb O139和毒力基因CT的特异性序列设计引物和TaqM an探针,建立优化TaqM an双重荧光PCR反应体系,进行特异性与敏感性的研究,并应用于模拟粪便感染和外环境监测标本的快速检测中。结果:该方法对产毒型O139群霍乱弧菌检测具有高度特异性,对rfbO139和CT基因序列检出限达到1.0×102cfu/m l或5 cfu/PCR反应体系,对模拟粪便感染和外环境监测标本的检测结果与实际情况完全一致,整个检测过程最快仅需3 h。结论:本研究建立的产毒型O139群霍乱弧菌双重荧光PCR检测技术具有特异性好、敏感性高、快速易操作等优点,可用于临床粪便标本和外环境监测样本的快速检测。
Objective: To study the establishment of TaqM an double fluorescent PCR technique for rapid detection of toxigenic O139 group Vibrio cholerae in simulated clinical specimens. Methods: Primers and TaqMa probes were designed based on the specific sequences of virulence gene CTV O139 gene of O139 cholerae O antigen and TaqMan probe. The optimized TaqM an double fluorescent PCR reaction system was established for specificity and sensitivity. And applied to simulate the rapid detection of fecal infection and external environmental monitoring specimens. Results: The method was highly specific for the detection of Vibrio cholerae O139 and the detection limit of rfbO139 and CT genes was 1.0 × 102cfu / ml or 5 cfu / PCR. The results showed that the detection limit of fecal infection and external environment Specimen test results and the actual situation exactly the same, the entire testing process as fast as 3 h. Conclusion: The double fluorescent PCR detection technique of Vibrio cholerae O139 produced in this study has the advantages of good specificity, high sensitivity, quick and easy operation and so on. It can be used in the rapid detection of clinical stool specimens and external environmental monitoring samples.