随机扩增多态性技术联合荧光定量PCR检测结核分枝杆菌方法的建立  

Establishment of random amplified polymorphic DNA combined with qPCR for detecting mycobacterium tuberculosis

作　　者：严玉娟 邢益平 施旭东[2] 严友德 艾月梅 刘雪 

机构地区：[1]南京医科大学第一附属医院感染科,江苏省210029 [2]南京市胸科医院检验科

出　　处：《江苏医药》2016年第11期1209-1213,共5页Jiangsu Medical Journal

基　　金：2011年江苏省医学创新团队基金项目(LJ201121)

摘　　要：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 develop a rapid and exact method to detect mycobacterium tuberculosis by combining the advantages of random amplified polymorphic DNA（RAPD）and fluorescence quantitative PCR（qPCR）.Methods According to the domestic and foreign literatures,a random primer for RAPD was designed.The reaction condition of RAPD was optimized using H37 Rv as a template and then 3distinct and bright bands appeared on the agarose gel electrophoretic plate.The DNA of 3bands was extracted,purified and processed for DNA sequencing.The results correlated with the ones published on NCBI and proved that the matching rate with mycobacterium tuberculosis reached 99%.One of them was choosen to design its specific primer and probe to process qPCR.The reaction condition of qPCR was optimized.The H37 Rv and other negative control bacteria were taken as templates to analyze the sensitivity and specificity of qPCR and RAPD-qPCR.Results qPCR could detect 30pg/μl of mycobacterium tuberculosis.Whereas RAPD-qPCR increased the sensitivity to3fg/μl.And the Ct values were lower[（6.58±0.19）～（14.95±0.36）]between 30pg/μl and30fg/μl of TB DNA in the samples by RAPD-qPCR,indicating that the results were stable and easy to be judged.The detection of all other clinically common bacteria was negative by both qPCR and RAPD-qPCR.Conclusion RAPD-qPCR is a new rapid and accurate method to detect mycobacterium tuberculosis.

关 键 词：结核分枝杆菌 随机扩增多态性技术 荧光定量聚合酶链式反应 

分 类 号：R378[医药卫生—病原生物学]