利用滚环扩增技术快速检测结核分枝杆菌rpoB基因单碱基突变

Detection of rpoB single base mutations in rifampin-resistant Mycobacterium tuberculosis by rolling circle amplification

目的应用滚环扩增(rolling circle amplification)技术建立对结核分枝杆菌耐药基因单碱基突变的快速检测方法。方法以结核分枝杆菌利福平耐药rpoB基因为研究对象,设计针对该基因常见突变位点的锁式探针。通过对突变型模板、野生型模板、非结核分枝杆菌基因序列模板及不同比例的突变型模板和野生型模板混合样本的检测,研究本实验方法的特异性和灵敏度。建立特异性地检测rpoB基因单碱基突变的滚环扩增方法。通过对临床样本的检测并与测序结果比较,评价该方法的临床应用价值。结果通过优化反应条件,应用滚环扩增技术能特异性的检测出结核分枝杆菌耐利福平rpoB基因的单碱基突变。通过对含有不同比例突变型模板的混合样本检测,最低能检测出1%突变型/野生型模板的样本。80株临床样本的检测与测序结果一致。结论滚环扩增技术特异性高、灵敏度高,无需特殊昂贵仪器且简单易操作,能够快速有效的检测出耐药结核的单碱基突变,是具有临床应用前景的方法。

Objective To establish a rapid detection method of single base mutations in rifampin-resistant Mycobacterium tuberculosis by rolling circle amplification （RCA）. Methods We used rpoB gene of rifampin-resistant Mycobacterium tuberculosis as the research target. Padlock probes were designed. The sensitivity and specificity of this method were evaluated by detecting mutation template, wild-type template, non-Mycobacteria tuberculous gene sequence template and different proportion of wild-type and mutation template mixed samples. The reaction conditions of RCA were optimized. The clinical values of the RCA method was evaluated by detecting the clinical samples and comparing the detection result with that of sequencing. Results RCA was used to detect the single base mutations of rpoB gene of rifampin-resistant Mycobacterium tuberculosis specifically. We proved that RCA method could detect 1% mutation template in different proportions of mixed wild-type and mutation template samples. The detection results of clinical samples were consistent with those of the sequencing. Conclusion There are many advantages of RCA, such as high specificity, high sensitivity, no special expensive instruments and simple operation. RCA is rapid in detection of single base mutations in rifampin-resistant Mycobacterium tuberculosis.