产毒型O139群霍乱弧菌特异性基因序列实时荧光双重TaqMan聚合酶链反应快速检测体系的建立  

Development of duplex real-time TaqMan PCR assay for detection of specific gene sequence from toxigenic Vibrio cholerae serogroup O139

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

机构地区：[1]北京协和医学院研究生院,北京100730 [2]卫生部北京医院卫生部老年医学研究所卫生部老年医学重点实验室,北京100730

出　　处：《中国卫生检验杂志》2009年第4期742-745,共4页Chinese Journal of Health Laboratory Technology

基　　金：国家高技术研究"863"发展规划(2002AA215013)

摘　　要：目的:建立针对O139群霍乱弧菌的高敏感、高特异的实时荧光双重TaqMan聚合酶链反应快速检测体系。方法:根据O139群霍乱弧菌主基因组O抗原编码基因rfb-0139和霍乱弧菌肠毒素A亚基的编码基因ctxA的特异性序列设计引物及探针,利用TaqMan标记探针和便携式Smartcycler II荧光实时PCR检测平台探讨该检测体系的检测敏感性,用19种其他肠道致病菌或院内感染中的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聚合酶链反应快速检测体系对O139群霍乱弧菌的检测敏感度为1.0×102拷贝每反应体系;对O139群霍乱弧菌基因组DNA的检测敏感度为1.0×100pg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均不存在非特异性扩增;整个反应在2 h内完成。结论:本研究建立的实时荧光双重TaqMan聚合酶链反应检测体系可作为O139群霍乱弧菌特异、敏感、快速的检测手段,并可同时检测O139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 the detection of toxigenic Vibrio cholerae O139. Methods：Designed primers and TaqMan probes for duplex PCR reaction using specific O antigen biosynthetic gene rib -O139 of serogroup O139 and cholera toxin gene ctxA as the target regions for amplification. Smart cycler system was used to evaluation the sensitivity of constructed assay method, and totally 19 other common enteropathogenic bacteria or frequent isolates causing nosocomial infection were also used to measure the specifity of constructed method. Results： The sensitivity of constructed assay method was 10^2 copies per reaction for the testing of fib - O139 gene. Using the Vibrio cholerae O139 genome DNA as the starting materials for detection, constructed assay method can detect as low as 1.0 × 10^0 pg per reaction. Non - specific amplifications were not presented when testing 19 other common enteropathogenic bacteria or frequent isolates causing nosocomial infection. In addition, the assay could get valid results within only 2 hours. Conclusion ：The duplex TaqMan real -time PCR assay described in this paper was so specific, sensitive and rapid that it was highly suitable not only for the discrimination of detection of Vibrio cholerae serogroup O139, but also for the detection of its virulent abilities simultaneously.

关 键 词：产毒型O139群霍乱弧菌 实时荧光TaqMan聚合酶链反应 霍乱肠毒素 

分 类 号：R378.3[医药卫生—病原生物学]