基于16SrDNA与CFP10基因的qPCR技术在脊柱结核诊断中的初步应用

发布时间：2018-01-27 07:11

本文关键词： 结核分枝杆菌DNA 脊柱结核荧光定量PCR 快速诊断 16Sr DNA CFP10　出处：《南华大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的探讨及推广荧光定量PCR技术在脊柱结核诊断中的应用方法选择结核分枝杆菌16Sr DNA和CFP10两个基因作为本次实验的靶基因,分别设计两对特异性的引物,应用SYBR Green I法(荧光染料法)建立荧光定量PCR反应体系,依据基因数据库中所提供的两种基因的保守序列,利用人工化学合成法获得目的基因,构建重组质粒体p GH-16Sr DNA和p GH-CFP10,并对重组质粒体进行酶切鉴定和序列测定,成功构建阳性标准品,并制作出标准曲线及曲线方程,最后用此方法对20例临床确诊为脊柱结核患者的血浆标本以及20例非脊柱结核患者的血浆标本进行检测与定量。结果本次实验中所建立的荧光定量方法线性关系好,均呈线性负相关。两个基因(16Sr DNA和CFP10)的标准曲线的相关系数分别达到0.999和0.998,实验中最低检测下限分别达到1.48×101copies/ul和2.67×101copies/ul,而且特异性好,两条基因的溶解曲线均呈现单一的峰值,没有出现非特异性扩增产物以及引物二聚体的干扰。在临床上已确诊的20例脊柱结核患者的血浆MTB-DNA的检测结果提示二者全部为阳性,20例非脊柱结核感染患者的血浆MTB-DNA检测结果有19为阴性,1例提示为阳性,并且平均检测一份临床血浆标本的时间仅为2小时。结论本实验成功建立了以16Sr DNA和CFP10为靶基因的脊柱结核荧光定量PCR检测体系,可以初步用于临床脊柱结核患者的结核分枝杆菌病原体检测及定量。
[Abstract]:Objective to explore and extend the application of fluorescent quantitative PCR in the diagnosis of spinal tuberculosis. Methods Mycobacterium tuberculosis 16Sr DNA and CFP10 genes were selected as the target genes in this experiment. Two pairs of specific primers were designed, and the fluorescent quantitative PCR reaction system was established by SYBR Green I method (fluorescent dye method). According to the conserved sequences of the two genes in the gene database, the target genes were obtained by artificial chemical synthesis, and the recombinant plasmids, p GH-16Sr DNA and p GH-CFP10, were constructed. The recombinant plasmid was identified by restriction endonuclease digestion and sequenced. The positive standard product was successfully constructed and the standard curve and curve equation were made. Finally, the plasma samples of 20 patients with clinically diagnosed spinal tuberculosis and 20 patients with non-spinal tuberculosis were detected and quantified by this method. Results the fluorescence quantitative method established in this experiment was linear. It's good. The correlation coefficients of the standard curves of 16Sr DNA and CFP10 were 0.999 and 0.998, respectively. The lowest detection limits were 1.48 脳 10 ~ (-1) copi / ul and 2.67 脳 10 ~ ((1)) / r ~ (-1) respectively, and the specificity was good. The dissolution curve of the two genes showed a single peak. There was no interference of nonspecific amplification products and primer dimer. The detection results of plasma MTB-DNA in 20 patients with spinal tuberculosis confirmed clinically showed that both of them were positive. The results of plasma MTB-DNA in 20 patients with non-spinal tuberculosis infection were 19 negative and 1 positive. The average time for detecting a clinical plasma sample was only 2 hours. Conclusion A fluorescent quantitative PCR system for detecting spinal tuberculosis with 16Sr DNA and CFP10 as target genes has been successfully established in this experiment. It can be used for the detection and quantification of Mycobacterium tuberculosis pathogens in patients with spinal tuberculosis.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R687.3

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