结核分枝杆菌聚合酶螺旋反应检测方法的建立及效果评价

Establishment and effect evaluation of polymerase spiral reaction for detection of Mycobacterium tuberculosis

目的建立聚合酶螺旋反应(polymerase spiral reaction,PSR)检测结核分枝杆菌的方法并评价效果。方法针对结核分枝杆菌插入序列IS6110设计引物进行PSR检测。提取结核分枝杆菌基因组DNA,加入Bst DNA聚合酶的RM2X反应液中,在荧光定量PCR仪上于63℃反应45 min,通过荧光信号进行检测。通过对引物序列进行优化,筛选出最佳引物序列,并对其检测特异性和最低检出限进行评估。于2019年5—8月间从郑州市第六人民医院连续收集200例肺结核患者痰液样本(同一患者收集3份),对痰标本进行痰涂片、固体痰培养和PSR检测。以痰培养结果为参照,评价PSR对结核病患者的检测效能。结果通过引物序列优化,筛选出一套对结核分枝杆菌检测的PSR引物,使用该引物进行PSR的最低检出限可达到10~3菌落形成单位(CFU)/ml;检测特异性实验表明该方法与15株非结核分枝杆菌无交叉反应。200例肺结核患者中,痰涂片检测阳性48例,阳性检出率为24.0%(48/200);痰培养检测阳性83例,阳性检出率为41.5%(83/200);PSR方法检测阳性87例,阳性检出率为43.5%(87/200)。以痰培养结果为标准,PSR检测结核病的敏感度和特异度分别为96.4%(80/83)、94.0%(110/117),阳性预测值为92.0%(80/87),阴性预测值为97.3%(110/113),与痰培养检测方法基本一致(Kappa值为0.898)。结论PSR检测适用于结核分枝杆菌的快速筛查。

Objective Establishment of polymerase spiral reaction(PSR)assay for detection of Mycobacterium tuberculosis(MTB)and evaluate its effect.Methods The PSR primers targeting the specific genes IS 6110 of MTB were designed.The genomic DNA of MTB was extracted and added into RM2×reaction solution containing Bst DNA polymerase.The reaction was carried out at 63℃for 45 min and detected by fluorescence quantitative PCR.By optimizing the primer sequence,the best one was selected,and its specificity and minimum detection limit were evaluated.A total of 200 sputum samples were collected from Zhengzhou Sixth Peoples Hospital during May 2019 to August 2019,and three copies were collected from the same patient.Sputum smear and sputum culture,as well as PSR detection were performed on sputum samples.Sputum culture was used as the gold standard to evaluate the effectiveness of PSR in detecting tuberculosis patients.Results A set of PSR primers for detection of MTB was screened by optimizing the primer sequence,the limit detection of PSR application to MTB was103 CFU/ml.The detection specificity experiment showed that the method had no cross-reactivity with 15 strains of nontuberculous mycobacteria.Among 200 pulmonary tuberculosis patients,48 cases were positive in sputum smears,and the positive detection rate was 24.0%(48/200);83 cases were positive in sputum culture test,and the positive detection rate was 41.5%(83/200);87 cases were positive in PSR test,and the positive detection rate was 43.5%(87/200).Compared with the gold standard method,the results of 200 clinical samples test showed that the sensitivity,specificity and positive predictive value,negative predictive value were 96.4%(80/83),94.0%(110/117),92.0%(80/87),97.3%(110/113),respectively,which were consistency to sputum culture detection method(Kappa value was 0.898).Conclusion PSR detection is suitable for the rapid diagnosis of MTB.