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目的以结核分枝杆菌的IS6110基因为特异性分子扩增靶标,利用TaqMan实时荧光定量PCR检测动物组织中结核分枝杆菌,以期作为科研或临床组织标本中结核分枝杆菌的检测方法。方法从155份小鼠、豚鼠和恒河猴的肺和脾组织中提取DNA,用TaqMan实时荧光定量PCR检测结核分枝杆菌IS6110基因,计算方法的灵敏度和特异度。结果实时荧光定量PCR对组织中结核分枝杆菌的最低检出限为1CFU/ml组织研磨液,其灵敏度和特异度分别为95.6%(95%CI:89.5%-98.4%)和100%(95%CI:89.6%-100%);相比而言,培养法的灵敏度和特异度分别为82.3%(95%CI:73.7%-88.6%)和100%(95%CI:89.6%-100%)。两种方法的kappa系数为0.762(95%CI:0.79-0.965)。结论 TaqMan实时荧光定量PCR能快速检测感染组织中的结核分枝杆菌,具有高灵敏度和特异度,适用于科研和临床实验室对组织标本中结核分枝杆菌的检测。
Objective To detect Mycobacterium tuberculosis in animal tissues by real-time TaqMan real-time fluorescence quantitative PCR with IS6110 gene of Mycobacterium tuberculosis as a target for the detection of Mycobacterium tuberculosis in scientific research or clinical tissue specimens. Methods DNA was extracted from the lung and spleen of 155 mice, guinea pigs and rhesus monkeys. The sensitivity and specificity of the method were tested by TaqMan real-time PCR. Results The detection limit of real-time PCR for detection of Mycobacterium tuberculosis in tissues was 1 CFU / ml. The sensitivity and specificity were 95.6% (95% CI: 89.5% -98.4%) and 100% (95% % CI: 89.6% -100%); compared with 82.3% (95% CI: 73.7% -88.6%) and 100% (95% CI: 89.6% -100% ). The kappa coefficient for both methods was 0.762 (95% CI: 0.79-0.965). Conclusion TaqMan real-time PCR can rapidly detect Mycobacterium tuberculosis in infected tissues with high sensitivity and specificity. It is suitable for the detection of Mycobacterium tuberculosis in tissue samples in scientific research and clinical laboratories.