产毒型O1群霍乱弧菌特异性基因序列实时荧光双重TaqMan PCR快速检测体系的建立  

Development of a rapid duplex TaqMan real-time PCR assay for detection of specific gene sequence from toxigenic Vibrio cholerae serogroup O1

作　　者：杨辉[1] 龚成[2] 吴丽娟[2] 胡继红[3] 汪仕奎[2] 蔡剑平[2] 

机构地区：[1]北京协和医学院,100730 [2]卫生部北京医院/卫生部临床检验中心/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 [3]北京大学医学部病原生物学系,100191

出　　处：《中国医药生物技术》2009年第1期9-13,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：目的建立针对O1群霍乱弧菌的高敏感、高特异的实时荧光双重TaqMan聚合酶链式反应快速检测体系。方法根据O1群霍乱弧菌主基因组O抗原编码基因rfb-O1和霍乱肠毒素的A亚基编码基因ctxA的特异性序列设计引物及TaqMan探针,利用便携式SmartcyclerⅡ实时荧光PCR检测平台探讨该检测体系的敏感性,用19种其他肠道致病菌及院内感染常见致病菌评价该检测体系的特异性。结果实时荧光双重TaqMan聚合酶链式反应快速检测体系对O1群霍乱弧菌的检测敏感度为1.0×102拷贝每反应体系;对O1群霍乱弧菌基因组DNA的检测敏感度为1.0×10–1pg每反应体系;该检测体系在检测19种其他肠道致病菌或院内感染中的常见致病菌时不存在非特异性扩增;整个反应在2h内完成。结论本研究建立的实时荧光双重TaqMan聚合酶链式反应检测体系可作为产毒型O1群霍乱弧菌特异、敏感、快速的检测手段,也可同时检测O1群霍乱弧菌产生霍乱肠毒素的能力。Objective To develop a sensitive and specific duplex TaqMan real-time quantitative polymerase chain reaction （PCR） assay for detection of toxigenic Vibrio cholerae O1. Methods Designed primers and Taqman probes were used for duplex PCR reaction using specific O antigen biosynthetic gene rfb of O1 serogroup and cholera toxin gene ctxA as the target regions for amplification. Smart cycler system was used to evaluate the sensitivity of constructed assay method and a total of 19 other common enteropathogenic bacteria or common isolates causing nosocomial infection were also used to measure the specificity of constructed method. Results The sensitivity of constructed assay method was 10^2 copies per reaction for the test of rfb-O1 gene. Using the Vibrio cholerae O1 genome DNA as the starting materials for detection, the constructed assay method can detect as low as 1.0×10^-1 pg per reaction. Non-specific amplifications were absent when testing 19 other common enteropathogenic bacteria or frequent nosocomial isolates. In addition, the assay could yield valid results only within 2 hours. Conclusion The duplex TaqMan real-time PCR assay described here is so specific, sensitive and rapid that it is perfectly suitable not only for the discriminative detection of O1 serogroup of Vibrio cholerae, but also for the detection of its virulence simultaneously.

关 键 词：弧菌 霍乱 O1 DNA探针 聚合酶链反应 霍乱毒素 

分 类 号：R450[医药卫生—治疗学]