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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 mutation of drug resistance gene of Mycobacterium tuberculosis by rolling circle amplification. Methods Mycobacterium tuberculosis rifampin-resistant rpoB gene was used as the research object to design a lock probe targeting to the common mutation sites of this gene. The specificity and sensitivity of this method were studied by detecting the mutant template, wild-type template, non-tuberculous mycobacterium gene sequence template and different proportion of mutant template and wild-type template mixed samples. A rolling-loop amplification method was established to detect the single-base mutation of rpoB gene specifically. The clinical value of this method was evaluated by comparing with the sequencing results. Results By optimizing the reaction conditions, single base mutation of rpoB gene of rifampicin in Mycobacterium tuberculosis could be detected specifically by rolling circle amplification. A minimum of 1% of the mutant / wild-type template was detected by testing mixed samples containing mutant mutants at different ratios. 80 clinical samples of the same test and sequencing results. Conclusion The technique of rolling ring amplification is of high specificity, high sensitivity, simple and easy operation without special and expensive instruments, and rapid and effective detection of single-base mutation of drug-resistant TB. It is a promising method for clinical application.