O1群霍乱弧菌实时荧光定量PCR快速检测方法的建立

Development of a Rapid TaqMan Real-Time PCR Assay for Detection of Vibrio cholerae Serogroup O1

目的:建立针对O1群霍乱弧菌的实时荧光定量TaqMan PCR快速检测方法,并进行模拟粪便标本的检测评价。方法:根据O1群霍乱弧菌O抗原编码基因rfb的特异性序列设计引物和TaqMan探针,建立检测O1群霍乱弧菌的实时荧光定量TaqMan PCR快速检测方法,对所建立的方法分别进行实验室内的灵敏度及特异性评价;将O1群霍乱弧菌灭活菌株悬液倍比稀释后与健康成人新鲜粪便混匀,制备成模拟带菌者粪便标本,提取DNA,进行Taq-Man PCR检测,用以评价该方法。结果:建立了快速检测O1群霍乱弧菌的实时荧光定量TaqMan PCR方法,灵敏度为每反应体系104拷贝;该方法对其他14种肠道菌DNA没有扩增;该方法对模拟粪便标本的检测灵敏度为每反应体系102 CFU。结论:建立了一种快速、高效检测O1群霍乱弧菌的荧光定量PCR检测方法,该方法可用于O1群霍乱弧菌临床粪便标本的检测。

Objective： To develop a TaqMan real-time quantitative PCR assay for detection of Vibrio cholerae serogroup O1,and to evaluate its reliability through detection of simulated cholerae carrier stool samples.Methods：O antigen rfb gene specific for V.cholerae serogroup O1 was used for the design of PCR primers and TaqMan probe,then the TaqMan PCR system in detecting V.cholerae serogroup O1 was developed,and its sensitivity and specificity were evaluated.A series of inactivated V.cholerae serogroup O1 organisms of known concentration were diluted to ten-folds continuously and mixed with fresh stool from healthy adult.Then it was used as simulated cholerae carrier stool samples spiked with V.cholerae,and was used to assess the assay.Results： The sensitivity of constructed assay method was 104 copies per reaction for the test of rfb-O1 gene.No amplification was observed in the templates of other 14 enteropathogenic bacteria.The sensitivity of the rapid TaqMan real-time PCR assay to detect the simulated cholerae carrier stool samples,was 1.0×102 CFU.Conclusion： The TaqMan real-time PCR assay described here is specific,sensitive and rapid,it is suitable for the discriminative detection of V.cholerae serogroup O1,and this assay could be used to detect the clinical stool samples with V.cholerae serogroup O1.