结核分支杆菌荧光PCR试剂盒的研制及临床试验

Development and clinical trial of fluorescence PCR diagnostic kit to detect Mycobacterium tuberculosis

目的用荧光PCR(fluorescence PCR,F-PCR)方法研制结核分支杆菌(TB)DNA检测试剂盒,通过临床试验评价其性能,并与其他方法进行比较。方法F-PCR是PCR和荧光探针杂交技术结合所产生的PCR方法。由于采用完全闭管荧光检测,避免了PCR后处理导致的假阳性污染;又采用了荧光检测技术,可以提高检测的灵敏性。应用F-PCR检测了297份肺结核病人和249份非结核对照者的痰液标本,以改良罗氏培养法、金胺荧光染液涂片法、Abbott公司的LCx试剂盒检测作为对照。结果设计合成了TB F-PCR诊断试剂盒。检测阳性率49.1%,灵敏性89.2%,特异性98.8%,符合率93.6%。结论F-PCR在灵敏性上显著优于培养法和涂片法,与LCx试剂盒检测无显著差异。F-PCR试剂盒可以检测TB的真实感染情况,对于临床诊断和疗效考察有一定的指导意义。

Objective To develop a new diagnostic kit for Mycobacterium tuberculosis(TB) detection on the basis of fluorescence PCR (F-PCR) and conduct clinical trial of this kit for assessing its performance in comparison with other diagnostic methods. Methods We collected 546 clinical sputum samples from patients with phthisis and normal subjects to examine TB by way of F-PCR in parallel with examination with modified R.culture, auramine smear with fluorescence staining and LCx DNA diagnostic kit from Abbott for comparison. Results A clinical diagnostic kit for TB detection was developed with F-PCR, and clinical trial showed that its positivity rate was 49.1%, sensitivity 89.2%, specificity 98.8% and efficiency 93.6%. Conclusion F-PCR is obviously superior to culture method and smear method and comparable with LCx in terms of sensitivity, and can be used to monitor TB DNA in the secretions to facilitate clinical diagnose and therapeutic effects monitoring.