分子信标荧光定量PCR快速检测结核分枝杆菌方法的初步研究

目的建立分子信标荧光定量PCR快速检测结核杆菌方法,为临床结核病的诊断提供特异性强、灵敏度高、可标准化、自动化的检测方法。方法根据GenBank数据库公布的结核分枝杆菌IS6110基因的保守序列,设计引物与分子信标探针,建立结核分枝杆菌荧光定量PCR检测方法;以10种细菌作对照分析该方法的特异性与灵敏度;应用已建立的方法检测结核分枝杆菌,评价其应用价值。结果所建立的分子信标荧光定量PCR检测10种细菌,只有结核分枝杆菌有荧光信号,与其他细菌无交叉反应,4拷贝/PCR反应体系的结核分枝杆菌都能有效检出。对100株结核分枝杆菌的检测结果为阳性,单一检测时间仅需2h。结论分子信标荧光定量PCR检测方法快速、灵敏度高、特异性强,可用于快速诊断结核病,为结核病的诊断与治疗提供新的检测手段。

Development of a Molecular Beacon Real-time PCR for the Rapid Detection of Mycobacterium Tuberculosis

Objective To develop a molecular beacon real-time PCR for rapid detection of Mycobacterium tuberculosis.Method One set of primers was selected from the IS6110 gene in GenBank and the corresponding molecular beacon probe was designed.The specificity and sensitivity of the developed method were evaluated by tested with 10 different bacteria species.The developed assay were also applied to the diagnosis of tuberculosis.Results Only Mycobacterium tuberculosis strains possessing IS6110 gene generated fluorescent signals,and no cross reaction was observed with other 9 bacteria.The detection limit was 4 copies/PCR reaction.100 Mycobacterium tuberculosis strains were positive tested by Real-time PCR.Conclusion The established molecular beacon real-time PCR is a rapid,specific and sensitive method,and is a beneficial supplement of traditional methods for the tuberculosis diagnosis.