Zhao Yong, Li Guilian, Sun Chongyun, Li Chao, Wang Xiaochen, Liu Haican, Zhang Pingping, Zhao Xiuqin, Wang Xinrui, Jiang Yi, Yang Ruifu, Wan Kanglin, Zhou Lei
Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, P. R. China.
Beijing Key Laboratory of POCT for Bioemergency and Clinic (No. BZ0329), Beijing 100071, P. R. China.
PLoS One. 2015 Nov 24;10(11):e0143444. doi: 10.1371/journal.pone.0143444. eCollection 2015.
Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a "probe dropout" manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.
通过分析相关基因序列的变异，耐药结核分枝杆菌可通过核酸扩增技术快速诊断。在本研究中，开发了一种基于锁核酸（LNA）探针的实时荧光定量PCR检测方法，以鉴定结核分枝杆菌中与利福平（RFP）耐药相关的rpoB基因突变。设计了6个具有单碱基错配鉴别能力的LNA探针，以监测23种最常见的rpoB基因突变。使用这些探针以“探针缺失”方式（定量循环 = 0）鉴定目标突变；因此，该技术在突变检测方面表现出优越性。基于LNA探针的实时荧光定量PCR检测方法采用双管形式，每管包含3个LNA探针和1个内部扩增对照探针。通过评估12株常见非结核分枝杆菌，该检测方法对有或无RFP耐药性的结核分枝杆菌均显示出优异的特异性。通过进一步引入巢式PCR方法，结核分枝杆菌的检测限为10个基因组当量（GE）/反应。在对154株临床分枝杆菌分离株的盲法验证中，通过该检测方法正确检测出142/142（100%）。在这些分离株中，88/88（100%）被确定为对RFP敏感，52/54（96.3%）被鉴定为对RFP耐药。对2株未识别的RFP耐药菌株进行测序，发现其突变位于23个突变靶点范围之外。总之，本研究建立了一种灵敏、准确且低成本的基于LNA探针的检测方法，适用于四通道实时荧光定量PCR仪器。该方法可用于临床实验室诊断RFP耐药结核病。
