论文部分内容阅读
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 establish a rapid, sensitive and specific real-time fluorescent double TaqMan polymerase chain reaction (PCR) system for the detection of Vibrio cholerae O139. Methods: Primers and probes were designed according to the specific sequence of coding gene ctxA of O139 cholera topoisomerase (V) gene of Vibrio cholerae and Vibrio cholerae enterotoxin A subunit. The TaqMan-labeled probe and portable Smartcycler II fluorescence Real-time PCR detection platform to explore the detection sensitivity of the detection system, with 19 kinds of other enteric pathogens or nosocomial infections in the common pathogen evaluation of the specificity of the test system. Results: The real-time fluorescence double TaqMan polymerase chain reaction rapid detection system for Vibrio cholerae O139 detection sensitivity of 1.0 × 102 copies per reaction system; Vibrio cholera O139 genomic DNA detection sensitivity of 1.0 × 100pg per reaction System; The detection system in the detection of 19 kinds of other enteric pathogens or nosocomial infections in the common pathogens are not non-specific amplification; the entire reaction within 2 h to complete. Conclusion: The real-time fluorescence double TaqMan polymerase chain reaction assay system established in this study can be used as a specific, sensitive and rapid detection tool for Vibrio cholerae O139 group and simultaneously detect the ability of Vibrio cholerae O139 to produce cholera enterotoxin.