探针导向重组酶介导等温扩增法检测MTB rpoB基因突变的应用价值

doi:10.3969/j.issn.1000-6621.2020.05.012

摘要/Abstract

摘要：

目的建立可快速检测MTB rpoB基因516位点突变的探针导向重组酶介导等温扩增法(probe-directed recombinase amplification,PDRA)。方法设计4条探针[1条野生型探针(P-W)及3条突变型探针(P-GGC、P-GTC、P-TAC)]和1条共同的下游引物,分别构建TB-516-W、TB-516-GGC、TB-516-GTC和TB-516-TAC 4种检测体系,在39℃条件下恒温扩增40min。通过阳性阈值时间判定MTB rpoB基因516位点是否发生突变及其突变类型;通过同一探针检测对应的重组质粒确定该检测体系的敏感度;通过不同探针检测某一种重组质粒,根据阳性阈值时间确定该检测体系的特异度。应用建立的PDRA方法检测6株利福平耐药MTB菌株和35份MTB培养阳性的痰标本,并与一代测序结果进行比较。结果 4种检测体系检测对应重组质粒的敏感度为1000拷贝/μl。4种检测体系的特异度良好,探针与靶序列完全匹配时,阳性阈值时间早;探针与靶序列不完全匹配时,阳性阈值时间晚,具有稳定的阳性阈值时间差值,体系TB-516-W,TB-516-GGC,TB-516-GTC和TB-516-TAC的阳性阈值时间差值分别为7.0、5.8、10.0、7.0min。PDRA检测6株MTB利福平耐药菌株和35份MTB培养阳性痰标本的结果与测序结果一致。结论本研究初步建立了可应用于MTB利福平耐药rpoB基因516位点突变检测的PDRA方法,该方法检测敏感度高,特异度好,具有一定的实验室应用价值。

关键词: 核酸扩增技术, 重组酶, 结核分枝杆菌, 基因

Abstract:

Objective To establish a probe-directed recombinase amplification (PDRA) assay for rapid detection of 516 mutations in Mycobacterium tuberculosis (MTB) rpoB gene. Methods Four probes including one wild-type (P-W) probe and three mutant-type probes (P-GGC, P-GTC, P-TAC) and a common downstream primer were designed to construct four detection systems (TB-516-W, TB-516-GGC, TB-516-GTC, and TB-516-TAC), respectively. The isothermal amplification was performed at 39 ℃ for 40 min. The positive threshold time was used to determine the occurrence and type of mutation at 516 locus of the MTB rpoB gene. The sensitivity of the detection system was assessed by detecting the corresponding recombinant plasmid with the same probe. The specificity of the detection system was assessed by detecting the same recombinant plasmid with different probes. The established PDRA assay was used to detect 6 MTB rifampicin-resistant strains and 35 MTB culture-positive sputum specimens, and compared with the results of Sanger sequencing. Results The sensitivity of the four detection systems for corresponding recombinant plasmids was 1000 copies/μl. When the probe and the target sequence were completely matched, the positive threshold time was short; otherwise, the positive threshold time was long, with a stable difference in positive threshold time, suggesting the good specificity of the four detection systems. The positive threshold time differences of the systems TB-516-W, TB-516-GGC, TB-516-GTC and TB-516-TAC were 7.0, 5.8, 10.0 and 7.0 min, respectively. The PDRA detection results of 6 MTB-resistant strains and 35 MTB culture-positive sputum samples were consistent with the sequencing results. Conclusion This study has preliminarily established a PDRA assay that can be applied to detect the 516 mutations in rpoB gene of MTB. It has high detection sensitivity and good specificity, and therefore has certain laboratory application value.

Key words: Nucleic acid amplification techniques, Recombinases, Mycobacterium tuberculosis, Genes

李鑫娜, 申辛欣, 王瑞白, 段素霞, 张瑞卿, 王瑞欢, 白雪丁, 凡国豪, 王金荣, 高源, 陈子巍, 马学军. 探针导向重组酶介导等温扩增法检测MTB rpoB基因突变的应用价值[J]. 中国防痨杂志, 2020, 42(5): 481-488. doi: 10.3969/j.issn.1000-6621.2020.05.012

LI Xin-na, SHEN Xin-xin, WANG Rui-bai, DUAN Su-xia, ZHANG Rui-qing, WANG Rui-huan, BAI Xue-ding, FAN Guo-hao, WANG Jin-rong, GAO Yuan, CHEN Zi-wei, MA Xue-jun. Application value of probe-directed recombinase amplification assay in detecting MTB rpoB gene mutation[J]. Chinese Journal of Antituberculosis, 2020, 42(5): 481-488. doi: 10.3969/j.issn.1000-6621.2020.05.012

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