类志贺邻单胞菌实时荧光TaqMan PCR快速检测体系的建立

Novel real-time TaqMan PCR assay for detection of Plesiomonas shigelloides

目的建立针对类志贺邻单胞菌的高灵敏、高特异的实时荧光TaqMan聚合酶链式反应(PCR)快速检测体系。方法根据类志贺邻单胞菌23S rRNA基因的一段特异性序列设计引物及TaqMan探针,利用实时荧光PCR检测平台探讨该检测体系的灵敏度;用30种其他肠道致病菌及院内感染中常见的致病菌评价该检测体系的特异性。结果实时荧光TaqMan PCR快速检测体系对类志贺邻单胞菌重组质粒的检测灵敏度为1×102拷贝/反应体系;对类志贺邻单胞菌基因组的检测灵敏度为3×10-2pg/反应体系;该检测体系在检测30种其他肠道致病菌及院内感染中常见的致病菌时未出现特异性扩增,整个反应在2 h内完成。结论本研究建立的实时荧光TaqMan PCR检测体系可作为类志贺邻单胞菌灵敏、特异、快速的检测方法。

Objective To develop a sensitive and specific real-time TaqMan polymerase chain reaction （PCR） assay for the detection of Plesiomonas shigelloides. Methods One set of primer and probe was designed based on the specific sequences of the 23S rRNA, and 7500 real-time PCR system was used to evaluate the sensitivity of the assay, and the specificity was evaluated by using 30 common enteropathogenic bacteria and some isolates causing nosocomial infection. Results The sensitivity of the real-time PCR assay was 1 x 102 copies per reaction for testing recombinant plasmids and 3 ×10^2pg per reaction for testing Plesiomonas shigelloides genome DNA. No specific amplifications were presented when the 30 common enteropathogenic bacteria and some isolates causing nosocomial infection were tested. Furthermore, the assay could be finished within 2 hours. Conclusion The real-time TaqMan PCR assay developed in our study was sensitive, specific and rapid, which could be used for the detection and isolation of Plesiomonas shigelloides.