荧光PCR熔解曲线法对痰样本结核分枝杆菌耐药性检测价值及耐药特征分析

目的：评估荧光PCR熔解曲线法（熔解曲线法）检测肺结核患者涂阳痰样本结核分枝杆菌（MTB）对一线抗结核药物耐药性价值及耐药特征分析。方法：收集涂阳且培养阳性肺结核患者痰样本和相应MTB菌株各142例，采用熔解曲线法检测痰样本MTB对抗结核药物链霉素（SM）、异烟肼（INH）、利福平（RFP）、乙胺丁醇（EMB）耐药情况，同时采用液体药敏法对相应MTB菌株耐药性检测，对两种检测结果行方法学比较。结果：以液体药敏法为标准，熔解曲线法检测痰样本MTB对4种抗结核药物耐药性结果差异无统计学意义（P>0.05），熔解曲线法检测SM、INH、RFP、EMB耐药性的敏感度分别为：78.57% (11/14)，70.00% (14/20)，100.00% (12/12)，71.43% (5/7)；特异度分别为：92.97%(119/128)，96.72%(118/122)，97.69%(127/130)，100%(135/135)；两者符合率分别为：91.55%(130/142)，92.96%(132/142)，97.89%(139/142)，98.59%(140/142)；Kappa值分别为：0.601，0.696，0.877，0.826。结论：与传统液体药敏检测相比，熔解曲线法检测痰样本MTB对抗结核药物SM、INH、RFP、EMB的耐药性效能一致，特异度和符合率均较高。该法对结核患者涂阳痰样本MTB耐药性检测有较好临床参考价值。

Fluorescent PCR melting curve method for detection of drug resistance of Mycobacterium tuberculosis in sputum samples and analysis of drug resistance characteristics

Objective: To evaluate the value of fluorescent PCR melting curve method (melting curve method) for detecting the anti-tuberculosis drug resistance of Mycobacterium tuberculosis in smear-positive sputum samples of tuberculosis patients and analysis of drug resistance characteristics. Methods: Collect sputum samples and corresponding isolated strains of tuberculosis patients with smear positive and culture-positive, and conduct Mycobacterium tuberculosis identification, obtain MTB-positive sputum samples and corresponding strains of 142 cases, and the melting curve method was used to detect Mycobacterium tuberculosis anti-tuberculosis drugs streptomycin (SM), isoniazid (INH), and rifampicin (RFP). ) And ethambutol (EMB) resistance mutations, while using traditional liquid drug susceptibility method to test the same anti-tuberculosis drug resistance of clinical isolates with positive sputum samples, and compare the two test results. Results: Compared with the liquid culture drug susceptibility method, the melting curve method for detecting the resistance of Mycobacterium tuberculosis in sputum samples to the four anti-tuberculosis drugs showed no significant difference (P>0.05). The liquid culture drug susceptibility method was used as the gold standard. The sensitivity of the curve method to detect the resistance of SM, INH, RFP, and EMB are: 78.57%,70.00%,100.00%,71.43%; the specificities are: 92.97%,96.72%, 97.69%, 100%; for both Consistency analysis of the test results of the two methods, the coincidence rates were: 91.55%, 92.96%, 97.89%, 98.59%; Kappa values were: 0.601, 0.696, 0.877, 0.826; indicating that the two detection schemes are effective for tuberculosis scores The test results of mycobacterial resistance are consistent. Conclusions: Compared with traditional liquid drug susceptibility testing, the fluorescence PCR melting curve method has the same efficacy in detecting the drug resistance of Mycobacterium tuberculosis in sputum samples to the anti-tuberculosis drugs SM, INH, RFP, and EMB, with higher sensitivity, specificity and coincidence rate. The melting curve method can be directly used for the detection of clinical sputum samples. It is fast, significantly shortens the detection time of drug-resistant Mycobacterium tuberculosis, and has good clinical reference value for the detection and treatment of drug-resistant tuberculosis.