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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 kit for detection of Mycobacterium tuberculosis (TB) DNA by fluorescence PCR (F-PCR) and evaluate its performance by clinical tests and compare with other methods. Method F-PCR is a PCR method that combines PCR and fluorescent probe hybridization techniques. Due to the completely closed fluorescent detection, the false positive contamination caused by PCR post-processing is avoided; and the fluorescence detection technology is adopted to improve the detection sensitivity. Sputum samples of 297 pulmonary tuberculosis patients and 249 non-tuberculosis controls were detected by F-PCR. The modified sputum samples were cultured by modified Roche culture method, gold amine fluorescence staining and Abbott LCx kit. Results The TB F-PCR diagnostic kit was designed and synthesized. The positive rate was 49.1%, the sensitivity was 89.2%, the specificity was 98.8% and the coincidence rate was 93.6%. Conclusion F-PCR was significantly superior to the culture method and smear method in sensitivity, which was not significantly different from LCx kit. F-PCR kit can detect the true infection of TB, for clinical diagnosis and efficacy of a certain significance.