外环境样本中产毒型O1群霍乱弧菌双重荧光定量PCR快速检测方法的建立

摘要

To establish a TaqMan-based multiplex real-time PCR assay for detection of toxigenic Vibrio cholerae serogroup O1 and construct a method for primary application of environmental samples test, the gene sequences of cholera toxin and specific O antigen biosynthetic gene rrb of serogroup O1 downloaded from the GenBank were aligned using the biologic software, and the specific primers and probe were designed in the conserved region of the CT and rfb-O1 gene for Vibrio cholerae serogroup O1. The reaction conditions were optimized and the sensitivity, specificity and the stability of the assay were evaluated. The clinical specimens collected from the environment were detected by this assay. For specifically detecting the toxigenic Vibrio cholerae serogroup O1, the detection limits of the assay for rfb-O1 and CT gene were 100 cfu/mL and the regression coefficient of the quantitative curve were 0. 998 and 0. 999, respectively. Five strains of toxigenic Vibrio cholerae serogroup O1 were collected from 352 environmental specimens for the first time by this assay. This assay is a rapid, sensitive and specific one for the widespread detection of toxigenic Vibrio cholerae serogroup O1 from environmental sample.%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.