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目的研究建立双重荧光定量PCR技术快速检测产毒型O1群霍乱弧菌,并首次应用于外环境样本的检测中。方法从GenBank上下载O1群霍乱弧菌O抗原编码基因rfbO1和毒力基因CT序列,在rfbO1和CT保守区域设计特异性引物和探针,建立优化单一和双重荧光PCR反应体系,评价所建双重PCR反应体系的特异性、敏感性和稳定性,并应用于外环境样本的监测检验中。结果该方法对O1群霍乱弧菌检测具有高度特异性,对rfbO1和CT基因序列检出限达到1.0×102cfu/mL,构建的体系定量标准曲线相关系数分别为0.999和0.998,具有较好的稳定性,并首次从352件外环境样本中检测出了5株产毒型O1群霍乱弧菌。结论本研究建立的双重荧光定量PCR方法特异、灵敏、快速,可应用于外环境样本中产毒型O1群霍乱弧菌的大范围筛查。
Objective To establish a dual-fluorescence quantitative PCR for the rapid detection of Vibrio cholerae O1-producing group O1 and for the first time applied to the detection of environmental samples. Methods The gene encoding rfbO1 and virulence genes of Vibrio cholerae O1 O1 were downloaded from GenBank. Specific primers and probes were designed in rfbO1 and CT conserved regions to establish optimized single and double fluorescent PCR reaction system. PCR reaction system specificity, sensitivity and stability, and applied to the monitoring of environmental samples in the test. Results The detection of Vibrio cholerae O1 was highly specific. The detection limit of rfbO1 and CT was 1.0 × 102 cfu / mL. The correlation coefficient of the quantitative standard curve was 0.999 and 0.998, respectively, which showed good stability For the first time, 5 strains of Vibrio cholerae O1 producing strains were detected from 352 environmental samples. Conclusion The double-fluorescence quantitative PCR method established in this study is specific, sensitive and rapid and can be applied to a wide range screening of Vibrio cholerae O1 producing group in external environment samples.