【摘要】：結核分枝桿菌（Mycobacterium Tuberculosis，MTB），是由德國科學家Koch于1882年發(fā)現(xiàn)，并證明其為結核病的病原體。隨著衛(wèi)生狀況的改善以及抗結核藥物的不斷發(fā)展，曾令結核病的發(fā)病率和病死率大幅度下降，然而80年代后，由于艾滋病（AIDS）的流行使結核病再度活躍。近年來，結核病的疫情呈現(xiàn)復蘇的趨勢，儼然成為傳染病中的頭號殺手和首要死亡病因。 利福平（RFP）是通過特異地與RNA聚合酶β亞基結合，從而抑制RNA聚合酶活性，并干擾分枝桿菌RNA的轉錄及合成，，最終阻礙蛋白質合成來發(fā)揮抗菌作用的。研究表明，90％以上結核桿菌耐RFP都是由于其作用的靶分子RNA多聚酶β亞單位的編碼基因（rpoB）發(fā)生突變所致。 目前檢測結核分枝桿菌主要依靠涂片、培養(yǎng)加藥敏、PCR技術等多項傳統(tǒng)試驗進行綜合分析，其操作復雜、耗時長，敏感以及特異性差，易導致誤診和漏診的發(fā)生，延誤治療的同時又加重了病人額外的經濟負擔。 本實驗采用反義抑制PCR的方法，檢測結核桿菌rpoB基因531以及526位點的突變，顯示當野生反義上游引物的濃度大于10μM時，可完全抑制野生型質粒DNA的PCR擴增；在此條件下，觀察到最低檢出濃度為1-5×103（IU/ml），其性能完全可以滿足臨床標本檢測的需要。 使用本方法對臨床182例結核病患者進行檢測，并與傳統(tǒng)的培養(yǎng)加藥敏法，以及PCR直接測序法結果進行比較。結果顯示本方法、藥敏法以及直接測序法所測得有rpoB基因突變分別為64例，63例，60例。本方法與直接測序法結果經卡方檢驗，統(tǒng)計學分析,結果顯示兩種方法檢測差異無統(tǒng)計學意義，說明本反義抑制PCR檢測方法檢測準確可靠。
[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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378.911

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