反义抑制PCR检测结核杆菌耐利福平变异菌株方法以及临床应用的研究

发布时间：2019-01-06 14:33

【摘要】：结核分枝杆菌（Mycobacterium Tuberculosis，MTB），是由德国科学家Koch于1882年发现，并证明其为结核病的病原体。随着卫生状况的改善以及抗结核药物的不断发展，曾令结核病的发病率和病死率大幅度下降，然而80年代后，由于艾滋病（AIDS）的流行使结核病再度活跃。近年来，结核病的疫情呈现复苏的趋势，俨然成为传染病中的头号杀手和首要死亡病因。利福平（RFP）是通过特异地与RNA聚合酶β亚基结合，从而抑制RNA聚合酶活性，并干扰分枝杆菌RNA的转录及合成，，最终阻碍蛋白质合成来发挥抗菌作用的。研究表明，90％以上结核杆菌耐RFP都是由于其作用的靶分子RNA多聚酶β亚单位的编码基因（rpoB）发生突变所致。目前检测结核分枝杆菌主要依靠涂片、培养加药敏、PCR技术等多项传统试验进行综合分析，其操作复杂、耗时长，敏感以及特异性差，易导致误诊和漏诊的发生，延误治疗的同时又加重了病人额外的经济负担。本实验采用反义抑制PCR的方法，检测结核杆菌rpoB基因531以及526位点的突变，显示当野生反义上游引物的浓度大于10μM时，可完全抑制野生型质粒DNA的PCR扩增；在此条件下，观察到最低检出浓度为1-5×103（IU/ml），其性能完全可以满足临床标本检测的需要。使用本方法对临床182例结核病患者进行检测，并与传统的培养加药敏法，以及PCR直接测序法结果进行比较。结果显示本方法、药敏法以及直接测序法所测得有rpoB基因突变分别为64例，63例，60例。本方法与直接测序法结果经卡方检验，统计学分析,结果显示两种方法检测差异无统计学意义，说明本反义抑制PCR检测方法检测准确可靠。
[Abstract]:Mycobacterium tuberculosis (Mycobacterium Tuberculosis,MTB) was discovered by German scientist Koch in 1882 and proved to be the pathogen of tuberculosis. With the improvement of health condition and the continuous development of anti-tuberculosis drugs, the incidence and mortality of tuberculosis have been greatly reduced. However, after the 1980s, the prevalence of (AIDS) made TB active again. In recent years, the epidemic of tuberculosis has shown a trend of recovery, has become the number one killer of infectious diseases and the leading cause of death. Rifampicin (RFP) inhibits the activity of RNA polymerase by specifically binding to RNA polymerase 尾 subunit, interferes with the transcription and synthesis of Mycobacterium RNA, and hinders protein synthesis to play an antibacterial role. The results showed that more than 90% of Mycobacterium tuberculosis resistance to RFP was due to the mutation of the encoding gene (rpoB) of its target molecule RNA polymerase 尾 subunit. At present, the detection of Mycobacterium tuberculosis mainly depends on smear, culture and drug sensitivity, PCR technology and other traditional tests for comprehensive analysis, its operation is complex, time-consuming, sensitive and poor specificity, easy to lead to misdiagnosis and missed diagnosis. Delays in treatment also add to the additional financial burden on patients. In this experiment, antisense inhibition of PCR was used to detect mutations at 531 and 526 of rpoB gene of Mycobacterium tuberculosis. The results showed that the PCR amplification of wild-type plasmid DNA could be completely inhibited when the concentration of wild antisense upstream primer was more than 10 渭 M. Under these conditions, the lowest detectable concentration was 1-5 脳 10 ~ 3 (IU/ml), and its performance could meet the needs of clinical specimen detection. This method was used to detect 182 cases of tuberculosis, and compared with the results of traditional culture and drug sensitivity method and PCR direct sequencing method. The results showed that the mutation of rpoB gene was detected in 64 cases, 63 cases and 60 cases by drug sensitivity assay and direct sequencing method, respectively. The results of chi-square test and direct sequencing showed that there was no significant difference between the two methods, indicating that the antisense inhibition PCR detection method was accurate and reliable.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378.911

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