以microRNAs为靶标结核病新型检测方法的建立

发布时间：2018-05-17 03:43

本文选题：微小RNA + 结核病　；参考：《吉林农业大学》2016年硕士论文

【摘要】：目的:目前全球性的传染性疾病有很多种,如艾滋病、病毒性肝炎、流行性感冒、结核病等,其中结核病是常见且严重危害人类健康的一种慢性传染性疾病,快速有效的早期诊断技术是结核病预防及治疗的前提。本研究以结核病人和健康人血清中的微小RNA(microRNAs)为研究对象,通过实时荧光定量PCR(qRT-PCR)技术检测结核病人与健康人血清中microRNAs的表达量,找到稳定差异表达的microRNAs分子,从而建立以microRNAs为检测靶标的新型结核病检测方法,以期开发出结核病的新型诊断技术。方法:本研究首先利用qRT-PCR方法检测了50例结核病人与50例健康人血清中与结核病高度相关的六种microRNAs分子(miR-29a,miR-93*,miR-320b,miR-144*,miR-21,miR-155)的表达量情况,找到稳定差异表达的有统计学意义的microRNAs分子。然后根据筛选结果,采用qRT-PCR方法对筛选出的稳定差异表达的microRNAs分子进行样本验证;同时利用酶联免疫吸附试验(ELISA)对样本进行检测,将两种方法的检测结果进行比较,讨论microRNAs分子是否有潜力作为结核病的新型生物诊断标志物应用于临床,从而评估以microRNAs为靶标的新型结核病检测方法的临床研发价值。结果:1.结核病人与健康人血清中miR-29a的表达量存在极显著差异;miR-320b的表达量存在显著差异;其它4种microRNAs(miR-93*,miR-21,miR-155,miR-144*)的表达量差异不显著。2.对miR-29a的荧光定量PCR反应体系进行优化,确定最佳模板浓度为5倍稀释的反转录cDNA;最适引物终浓度为0.2μM,建立了以miR-29a为靶标的结核病新型检测方法。3.以miR-29a为靶标的结核病检测方法阳性检测率为94%;ELISA方法的阳性检测率为89%。结论:结核病人与健康人血清中miR-29a的表达量存在极显著差异;建立了以miR-29a为靶标的结核病新型检测方法;该检测方法对结核病的检测效果较好。
[Abstract]:Objective: at present, there are many kinds of global infectious diseases, such as AIDS, viral hepatitis, influenza, tuberculosis and so on. Rapid and effective early diagnosis is the premise of tuberculosis prevention and treatment. In this study, microRNAs in the sera of TB patients and healthy people were used to detect the expression of microRNAs in the sera of TB patients and healthy people by real-time fluorescence quantitative PCRQ RT-PCRR technique, and stable differentially expressed microRNAs molecules were found. In order to develop a new diagnosis technique of tuberculosis, a new method of tuberculosis detection based on microRNAs was established. Methods: in this study, qRT-PCR method was used to detect the expression of six microRNAs molecules (miR-29a1 miR-93), which are highly correlated with tuberculosis, in the sera of 50 TB patients and 50 healthy controls. The stable differentially expressed microRNAs molecules were found. Then, according to the screening results, the qRT-PCR method was used to verify the samples of the stable differentially expressed microRNAs molecules, and the Elisa assay was used to detect the samples, and the results of the two methods were compared. This paper discusses the potential of microRNAs molecule as a new biomarker for diagnosis of tuberculosis, so as to evaluate the value of clinical research and development of new TB detection method targeting microRNAs. The result is 1: 1. There was a significant difference in the expression of miR-29a between tuberculosis patients and healthy people, but there was no significant difference in the expression of miR-155miR-144b among the other four microRNAsmiR-93s. The fluorescence quantitative PCR reaction system of miR-29a was optimized to determine the best template concentration of 5 times dilution of reverse transcriptional cDNAand the optimal final concentration of primers was 0.2 渭 M. A new detection method of tuberculosis targeting miR-29a was established. The positive rate of TB detection method based on miR-29a was 94%. The positive rate of Elisa method was 89%. Conclusion: there is a significant difference in the expression of miR-29a between tuberculosis patients and healthy people, and a new detection method for tuberculosis, which is targeted at miR-29a, has been established, which has a good effect on the detection of tuberculosis.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R52;R440

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