产毒型霍乱弧菌实时定量三重TaqMan PCR快速检测方法的建立

Development of a rapid triplex TaqMan real-time PCR assay for the detection of toxigenic Vibrio cholerae

目的研究建立可同时检测产毒型O1群及O139群霍乱弧菌的实时荧光三重TaqManPCR快速检测方法。方法根据O1群和O139群霍乱弧菌主基因组O抗原编码基因的特异性序列rfb-O1和rfb-O139，以及霍乱弧菌肠毒素A亚基的编码基因ctxA序列设计引物及探针，利用TaqMan标记探针和便携式SmartcyclerII实时荧光PCR检测平台探讨该三重检测方法的检测敏感性，并用19种其他肠道致病菌或院内感染中的常见致病菌评价了该检测方法的特异性。结果实时荧光三重TaqManPCR快速检测方法对01群及0139群霍乱弧菌的检测灵敏度均为每反应体系1．0×10^2拷贝，线性检测范围为每反应体系1．0×10^2～1．0×10^2拷贝；对01群霍乱弧菌基因组DNA的检测灵敏度为每反应体系1．0×10～pg，对0139群霍乱弧菌基因组DNA的检测灵敏度为每反应1pg，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三重TaqManPCR检测方法可作为鉴别产毒型O1群及O139群霍乱弧菌特异、敏感、快速的检测手段，也可同时检测相应霍乱弧菌产生霍乱毒素的能力。

Objective To develop a rapid triplex TaqMan real-time quantitative polymerase chain reaction （PCR） assay for the discrimination of toxigenic Vibrio cholerae O1 and Vibrio cholerae O139 simuhaneously. Methods Primers and Taqman probes were designed for multiplex PCR reaction using rJS-O1, rfb-O139 specific O antigen biosynthetic gene and cholera toxin gone ctxA as the target regions for amplification. Smart cycler system was used to evaluate the sensitivity of this method, and a total of 19 other common enteropathogenic bacteria or frequent isolates causing nosocomial infection were also used to measure the specificity of constructed method. Results Constructed assay method allowed the detection as few as 102 copies per reaction for the testing of ctxA, 9rb-O1 and rfb-O139 genes using mixed plasmids as the templates, and the linearity range of assay varied from 102 to 109 copies per reaction. Using the Vibrio cholerae O1 and O139 genome DNAs as the starting materials for detection, constructed assay method could detect I. 0 x 10-1 pg and 1.0 x 10~ pg per reaction, respectively. 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 triplex TaqMan real-time PCR assay described in this study was specific, sensitive,rapid and suitable not only for the discrimination of toxigenic strains,O1 and O139 serogroup of Vibrio cholerae, but also for the detection of their virulent abilities simultaneously.