基于双启动寡聚核苷酸引物的沙门菌、志贺菌、弯曲杆菌、耶尔森菌多重实时荧光PCR方法的建立

Establishment of multiplex real-time fluorescence PCR for Salmonella,Shigella,Campylobacter and Yersinia enterocolitica basing on dual priming oligonucleotide system

目的建立多重实时荧光PCR同时检测沙门菌、志贺菌、弯曲杆菌、耶尔森菌的方法。方法通过设计针对沙门菌、志贺菌、弯曲杆菌和耶尔森菌的双寡聚核苷酸引物,来建立这4种病原体的多重PCR检测体系。结果建立的多重PCR体系可同时特异性扩增4种病原体的DNA片段。沙门菌和弯曲杆菌的特异性和敏感度都是100.0%,志贺菌和耶尔森菌的敏感度是100.0%,特异性分别是99.3%和98.6%。最低检测限均为103 copies/mL。结论该体系可快速、有效、准确检测出样品中4种病原体的存在,可用于肠胃道相关病原体感染的辅助评价。

Objective To establish a multiplex real-time fluorescence PCR assay for simultaneously detecting Salmonella,Shigella,Campylobacter and Yersinia enterocolitica.Methods Dual priming oligonucleotide（DPO）was designed for these four pathogens.A multiplex RT-PCR assay system was developed using DPO primers.ResultsThe specific DNA fragments of 4kinds of pathogen were amplified simultaneously and specifically by this established PCR assay system.The specificity and sensitivity of the assay for detecting Salmonella and Campylobacter all were100.0%,the sensitivities for detecting Shigella and Yersinia enterocolitica were 100.0% and the specificities were99.3% and 98.6% respectively.The limit of detection（LOD）was 103 copies/mL.Conclusion This multiplex RTPCR system can detect the existence of these four kinds of pathogen rapidly,effectively and accurately and can be used the auxiliary evaluation of gastrointestinal related pathogen infection.