TaqMan-MGB荧光定量PCR快速检测痰结核分枝杆菌异烟肼耐药效果的评价

目的评价通过检测结核分枝杆菌katG基因突变以诊断异烟肼耐药并检测结核分枝杆菌的TaqMan-MGB荧光定量PCR方法。方法收集临床诊断为肺结核病的患者痰样本,用已建立的荧光PCR方法进行检测,与常规痰涂片抗酸染色、分枝杆菌培养和传统比例法药敏试验比较,评价检测结核分枝杆菌敏感性和特异性及异烟肼耐药的特异性和敏感性。统计学分析采用χ2检验,P值<0.05为差异有统计学意义。结果荧光定量PCR共检测186份痰液样本,敏感性为84.47%,特异性为100.00%。在确诊病人的痰液中阳性检出率84.47%,明显高于痰涂片40.99%(χ2=46.44,P<0.01),痰培养40.37%(χ2=47.89,P<0.01),且差异有统计学意义。其中荧光PCR在菌阳痰液中检出率94.44%,涂阴培阴的痰液中检出率75.28%。与常规药敏试验结果比较,培养阳性的61份痰样本的药敏符合率96.72%(59/61),敏感性33.33%(1/3),特异性100.00%(58/58)。结论 TaqMan-MGB荧光定量PCR的方法能特异、灵敏、快速诊断痰液中的结核分枝杆菌及其异烟肼耐药性。

Study on rapid diagnosis of Mycobacterium tuberculosis resistance to isoniazid from sputum with TaqMan-MGB real-time PCR

Objective To evaluate a TaqMan-MGB real-time PCR assay for detecting the katG gene mutation to diagnose Mycobacterium tuberculosis(M.tuberculosis) resistance to Isoniazid(INH).MethodsThe sputum samples were collected from the patients with tuberculosis(TB) for detecting M.tuberculosis by the TaqMan-MGB real-time PCR,at the same time sputa smear microscopy,sputa smear microscopy and conventional drug sensitivity test(DST) were used as controls.The sensitivity and specificity of the TaqMan-MGB real-time PCR for detecting M.tuberculosis and the resistance to INH were evaluated.The statistical analysis of the results was performed by χ2 test,and P value<0.05 was considered as statistical significance.ResultsFor detection of 186 sputum samples,the sensitivity and specificity of the real-time PCR assay were 84.47% and 100% respectively.For detecting the sputa of patients confirmed with TB,the positive rate of the real-time PCR was 84.47%,significantly higher than that of the sputa smear microscopy(40.99%,χ2=46.44,P<0.01) and the culture with L-J medium(40.37%,χ2=47.89,P<0.01).The positive rate of the real-time PCR was 94.44% in the positive samples tested by smear microscopy or culture method,whereas it was 75.28% in the negative sputum samples.Compared with the results of conventional drug sensitivity test(DST) to detect INH-resistance with 61 culture-positive sputum samples,the concordance rate was 96.72%(59/61) and the sensitivity and specificity of this method were 33.33%(1/3) and 100%(58/58) respectively.ConclusionsThe method of TaqMan-MGB real-time PCR could detect katG 315 G→C mutation of M.tuberculosis in clinical sputum specifically,sensitively and rapidly to use the diagnosis of TB and M.tuberculosis drug resistance to INH simultaneously.