改良分子信标-实时PCR快速检测志贺菌

Application of modified molecular beacon based real-time PCR assay for detection of Shigella

目的:建立改良分子信标-实时PCR检测志贺菌的快速方法,应用于志贺菌食物中毒的快速诊断和门诊肠道致病菌的检测。方法:根据GenBank公布志贺菌ipaH基因的保守序列,设计引物和改良分子信标探针,建立实时PCR-改良分子信标检测体系,应用于对志贺菌食物中毒的快速诊断和门诊肠道致病菌的检测。结果:改良分子信标-实时PCR反应体系DNA灵敏度为93 fgμ/l,菌液灵敏度为64 cfu/m l或2 cfu/PCR反应体系,无交叉反应。此反应体系检测67株志贺菌,均出现特异的荧光信号。对细菌性食物中毒样本等共657份样品进行志贺菌检测,42份志贺菌实时PCR阳性,其中41份志贺菌细菌培养阳性。从样品处理到检测结果仅需2 h^1 d时间。结论:改良分子信标-实时PCR检测体系快速、灵敏度高,特异性强,可用于志贺菌食物中毒的快速诊断,为食源性疾病的分子流行病学调查提供新的检测手段,对于提高肠道门诊的工作效率有重要意义。

Objective：Rapid detection assay of Shigella using modified molecular beacon and real -time PCR was developed. The established method was applied to rapid diagnosis of Shigella food poisoning, and health examination. Methods： Based on the core sequence of ipaH gene of Shigella published on GenBank, one set of primers and the corresponding modified molecular beacon were designed. The modified molecular beacon was labeled with FAM. Then the molecular beacon and primers were tested against 22 different bacterial species. Finally the real - time PCR assay was applied to the rapid diagnosis of food poisoning and disease surveillance. Results： For the modified molecular beacon based real -time PCR assay, the sensitivity was 93 fg/μl or 2 cfu/PCR reaction. There was no cross - reaction with other bacteria as control. The real - time PCR assay was used to detect 67 Shigella strains and no false signals were observed. Among 657 samples, 42 were positive for real time PCR and 41 were positive for conventional method. The overall test could be finished within one day starting from sample preparation. Conclusion：The modi- fied molecular beacon based real time PCR assay is rapid, sensitive, and specific. It could be applied to the rapid diagnosis of Shigella food poisoning and health examination.