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目的结合随机扩增多态性技术(RAPD)和荧光定量聚合酶链式反应技术(qPCR)的优势,建立快速、准确地检测结核分枝杆菌的新方法。方法根据国内外文献设计随机引物,以结核分枝杆菌标准菌株H37Rv为模板进行RAPD,产物进行琼脂糖凝胶电泳,选取条带进行割胶纯化测序,在美国国家生物技术信息中心(NCBI)的基本局部比对搜索工具(BLAST)程序上进行同源性检索,结果和结核分枝杆菌匹配率都达到99%。设计特异性内引物和探针,以RAPD产物为模板进行qPCR,并优化反应条件。分别以10倍梯度稀释的一系列浓度的H37Rv和其他阴性对照菌为模板,进行qPCR和RAPD-qPCR,分析其灵敏性和特异性。结果 qPCR能够检测到30pg/μl的结核分枝杆菌,而RAPD-qPCR灵敏性提高到3fg/μl。而且RAPD-qPCR检测30pg/μl至30fg/μl的结核分枝杆菌核酸时,扩增得到的Ct值都较小[(6.58±0.19)~(14.95±0.36)],结果稳定,易于判断。qPCR和RAPD-qPCR检测其他临床常见的细菌均呈现阴性。结论 RAPD-qPCR是一种快速、准确地检测出结核分枝杆菌的新方法。
Objective To establish a rapid and accurate method for the detection of Mycobacterium tuberculosis by combining the advantages of random amplified polymorphic (RAPD) and quantitative polymerase chain reaction (qPCR). Methods Random primers were designed according to domestic and international literatures, RAPD was carried out using Mycobacterium tuberculosis standard strain H37Rv as a template, and the products were subjected to agarose gel electrophoresis. The bands were selected and cut for gel purification. Sequencing was performed at the National Basic Information Center for Biotechnology (NCBI) Homology search was performed on the BLAST program with a 99% match with M. tuberculosis. Specific primers and probes were designed, qPCR was performed using RAPD products as template, and the reaction conditions were optimized. The sensitivity and specificity of qPCR and RAPD-qPCR were analyzed using a series of 10-fold dilutions of H37Rv and other negative control bacteria as templates. Results qPCR was able to detect 30pg / μl of M. tuberculosis, while RAPD-qPCR sensitivity was increased to 3fg / μl. Moreover, when RAPD-qPCR detected 30pg / μl to 30fg / μl of Mycobacterium tuberculosis nucleic acid, the Ct values obtained by amplification were all smaller ([6.58 ± 0.19] ~ (14.95 ± 0.36)]. The results were stable and easy to judge. qPCR and RAPD-qPCR detection of other clinical common bacteria were negative. Conclusion RAPD-qPCR is a new method for rapid and accurate detection of Mycobacterium tuberculosis.