产毒型O1群霍乱弧菌实时荧光双重TaqMan PCR的临床应用前研究  

Pre-clinical application research on a rapid duplex TaqMan real-time PCR assay for the detection of toxigenic Vibrio cholerae serogroup O1

作　　者：胡继红[1] 杨辉[2] 吴丽娟[3] 龚成[3] 戴大鹏[3] 蔡剑平[3] 庄辉[1] 

机构地区：[1]北京大学医学部病原生物学系,100191 [2]北京协和医学院,100730 [3]卫生部北京医院/卫生部北京老年医学研究所/卫生部老年医学重点实验室,100730

出　　处：《中国医药生物技术》2009年第1期14-18,共5页Chinese Medicinal Biotechnology

基　　金：国家高技术研究发展计划(863计划)(2002AA215013)

摘　　要：目的探讨产毒型O1群霍乱弧菌实时荧光双重TaqMan PCR法对临床模拟标本检测的意义,为该法的临床应用提供可靠的技术依据。方法将O1群霍乱弧菌灭活菌株悬液[1.0×1011CFU(菌落形成单位)/ml]连续10倍稀释后与健康成人新鲜粪便混匀,制备成5.0×101～5.0×109CFU/g梯度浓度的模拟带菌者粪便标本并提取DNA,用以评价产毒型O1群霍乱弧菌实时荧光双重TaqMan PCR法对临床模拟标本检测的灵敏度、特异性和稳定性。结果实时荧光双重TaqMan PCR法对O1群霍乱弧菌的模拟带菌者粪便标本的检测灵敏度为1.0×103CFU(5.0×104CFU/g)每反应体系,其检测线性范围为1.0×103～1.0×108CFU每反应体系;在稳定性评价中,对含菌量1.0×103～1.0×108CFU每反应体系模拟带菌者粪便标本DNA进行3次重复检测的结果显示,ctxA基因扩增反应Ct值的变异系数为0.54%～1.36%,rfb-O1基因扩增反应Ct值的变异系数为1.65%～3.75%;在特异性评价中,3名健康成人新鲜粪便标本的阴性对照均未见特异性扩增曲线。结论产毒型O1群霍乱弧菌实时荧光双重TaqMan PCR法可用于O1群霍乱弧菌临床粪便标本的检测。Objective To evaluate the significance of a rapid duplex TaqMan real-time PCR assay for detection of toxigenic Vibrio cholerae serogroup O 1, and providing a reliable technical basis for its clinical application. Methods A series of inactivated Echolerae serogroup O1 organisms of known concentration （1.0×10^11 CFU/ml） were diluted to ten-folds continuously and mixed with fresh stool from healthy adult. Then it was used as simulated cholerae cartier stool samples spiked with Echolerae of 5.0 ×10^1 - 5.0×10^9 CFU/g ladder concentration. The simulated samples were used to assess the sensitivity, specificity and stability of the duplex TaqMan real-time PCR assay for the detection of toxigenic Vibrio cholerae serogroup O1. Results The sensitivity of the rapid duplex TaqMan real-time PCR assay was 1.0×10^3 CFU （5.0×10^4 CFU/g）, and the linear range was 1.0×10^3 - 1.0×10^8 CFU. Three groups of different V. cholerae concentrations of simulated samples were tested for three times, the CV% for Ct value of detecting ctxA gene and rfb-O1 gene were 0.54% - 1.36% and 1.65% - 3.75%, respectively. No specific amplification was found in the negative control samples collected from the healthy adult stools without E cholerae. Conclusion The rapid duplex TaqMan real-time PCR assay may be used as one sensitive, specific and stabile method for detecting clinical stool samples with toxigenic Vibrio cholerae serogroup O 1.

关 键 词：弧菌 霍乱 O1 聚合酶链反应 霍乱毒素 

分 类 号：R450[医药卫生—治疗学]