【摘要】：背景研究 結(jié)核性腦膜炎由結(jié)核桿菌引起的中樞神經(jīng)系統(tǒng)感染是最嚴重的肺外結(jié)核。雖然結(jié)核菌的耐藥率在結(jié)核性腦膜炎患者中明顯低于肺部感染患者，但是多耐藥結(jié)核性腦膜炎的患者會遺留更嚴重的神經(jīng)系統(tǒng)后遺癥或者直接死亡，給社會和家庭造成了嚴重的負擔�；颊叱狈焖贉蚀_的診斷和藥物敏感性檢測，而延誤治療，最終導致嚴重后遺癥、產(chǎn)生多耐藥結(jié)核或使多耐藥結(jié)核桿菌更進一步發(fā)展成為廣泛性耐藥，，使得結(jié)核控制產(chǎn)生了更嚴峻的挑戰(zhàn)。WHO推薦結(jié)核桿菌感染患者需要早期接受正規(guī)，足量治療，并且每個患者需接受藥物敏感性檢測性，并根據(jù)其耐藥診斷，選擇合理的藥物治療。規(guī)范的治療在肺結(jié)核治療中降低致殘率和致死率并且提高預后和控制多耐藥結(jié)核的發(fā)生率等方面都起到了明顯的作用。但是目前結(jié)核性腦膜炎治療中，由于沒有明確的診斷和治療指南，往往是根據(jù)肺結(jié)核的診治方法及經(jīng)驗用藥。且由于血腦屏障等因素，往往用藥劑量和時程均大于肺結(jié)核，例如在結(jié)核性腦膜炎的經(jīng)驗治療中利福平用藥比肺結(jié)核治療時，劑量大，時間長，沒有規(guī)范治療和用藥則更有可能導致利福平耐藥。但是由于結(jié)核性腦膜炎病例很難收集，腦脊液結(jié)核桿菌數(shù)量少，很難培養(yǎng)成功等現(xiàn)狀，針對結(jié)核性腦膜炎患者的耐利福平結(jié)核分枝桿菌基因突變等機制研究及結(jié)核性腦膜炎耐藥檢測方法研究并不充分，更無法做到詢證醫(yī)學。所以多耐藥結(jié)核性腦膜炎檢測及機制還有需要更多的研究支持。 目的 鑒于肺結(jié)核與結(jié)核性腦膜炎治療中利福平用藥劑量及治療時間上的差異，探究這兩種患者中的利福平耐藥的突變位點是否存在差異；建立一個快速，簡單，高通量，低成本的檢測方法可以檢測結(jié)核分枝桿菌耐藥突變基因，并用于結(jié)核性腦膜炎的診斷。 方法 通過測序的方法比較導致結(jié)核性腦膜炎的結(jié)核分枝桿菌利福平耐藥基因突變機制與致肺結(jié)核結(jié)核分支桿菌耐藥基因突變位點有無差別，并根據(jù)結(jié)核耐藥數(shù)據(jù)庫給予的結(jié)果，選取高度確信耐藥位點，通過NCBI的GENE數(shù)據(jù)庫獲得rpoB基因（Rv0667），根據(jù)M-ARMS-PCR原理進行設計引物，設計五對引物分別為1037OF-1048OR；511IF-1478OR；516IF-1478OR；526IF-1478OR；531IF-1478OR，其中5個單獨的上游引物，均與下游1478OR組成4對引物，分別形成半巢式PCR，產(chǎn)物大小分別：442bp、227bp、214bp、183bp、166bp。選取135例培養(yǎng)陽性并通過藥敏檢測的結(jié)核分支桿菌菌株，進行M-ARMS-PCR擴增，并應用QIAxcel儀器進行檢測分析。同時針對這135例菌株的耐藥決定區(qū)域進行DNA測序。應用藥敏結(jié)果和測序結(jié)果對M-ARMS-PCR方法進行評價。 結(jié)果 本次研究通過測序的方法對比導致結(jié)核性腦膜炎利福平耐藥的結(jié)核分枝桿菌與導致肺結(jié)核結(jié)核分枝桿菌利福平耐藥rpoB基因突變的分布情況，102株利福平耐藥的結(jié)核分枝桿菌的rpoB基因測序發(fā)現(xiàn)，63株導致肺結(jié)核的結(jié)核分枝桿菌的rpoB基因突變常見位點為：531（53.97%），526（20.63%），516（6.45%）。39株導致結(jié)核性腦腦膜炎的rpoB基因突變最常見位點為：531（61.54%），526（20.51%），533（7.69%）。建立了基于QIAxcel檢測平臺的利福平耐藥基因rpoB檢測的M-ARMS-PCR檢測方法。這種方法能夠單管5重選擇性PCR擴增rpoB基因高度確信耐藥四個密碼子511、516、526、531的野生型基因，不擴增突變型。M-ARMS-PCR結(jié)果中，131例樣品與DNA測序結(jié)果一致。用DNA測序方法評價M-ARMS-PCR方法的敏感度為94.2%，特異性為100%。藥敏檢測方法評價M-ARMS-PCR的方法的敏感度為86.57%，特異性為89.71%。 結(jié)論 通過本次試驗了解了導致結(jié)核性腦膜炎的結(jié)核分支桿菌利福平耐藥rpoB基因最常見的突變區(qū)域為531-533區(qū)域。其中531位密碼子的Ser-Leu突變率占明顯優(yōu)勢。 建立的基于QIAxcel檢測平臺的M-ARMS-PCR的檢測方法檢測利福平耐藥基因突變，可以在6個小時能完成96個樣品檢測達到了預期實驗目的。具有便捷，快速，準確，早期，廉價的優(yōu)點，適宜應用于貧困，醫(yī)療水平相對落后的地區(qū)。
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圖片說明：具有代表性TBM樣品測序峰圖
[Abstract]:background research The central nervous system infection caused by tuberculosis of tuberculous meningitis is the most serious extrapulmonary junction. Nuclear. Although the resistance rate of the tubercle bacillus is significantly lower than that of the patients with pulmonary infection in the patients with tuberculous meningitis, the patients with multi-drug-resistant tuberculous meningitis left more serious neurological sequela or direct death, causing serious negative effects on the society and the family The patients often lack rapid and accurate diagnosis and drug sensitivity detection, and delay treatment, resulting in serious sequela, multi-drug-resistant tuberculosis or even further development of the multi-drug-resistant mycobacterium tuberculosis, so that the control of the tuberculosis is more severe. The WHO recommends that patients with M. tuberculosis infection require early acceptance of normal, adequate treatment, and each patient is subject to drug sensitivity detection and, based on its drug resistance diagnosis, select a reasonable drug treatment The treatment has played an important role in the treatment of pulmonary tuberculosis, the reduction of the disability rate and the mortality rate, the improvement of the prognosis and the control of the incidence of multi-drug-resistant tuberculosis, etc. In the treatment of tuberculous meningitis, there is no clear diagnosis and treatment guide, often based on the diagnosis and treatment of pulmonary tuberculosis and experience. As a result of the blood-brain barrier and other factors, the dosage and time history of the drug are more than that of the pulmonary tuberculosis, for example, when the rifampin is used in the treatment of the tuberculosis meningitis, the dosage is large, the time is long, the treatment and the medication are not standardized, and the rifampin resistance is more likely to be caused. However, because of the difficulty in collecting the cases of tuberculous meningitis, the number of tubercle bacillus in the cerebrospinal fluid is small, it is difficult to culture the present status, and the research on the mechanism of the detection of the resistance to the rifampin-resistant Mycobacterium tuberculosis in the patients with tuberculous meningitis and the method of the detection of the drug resistance of the tuberculous meningitis do not charge. No, I can't make an inquiry. Therefore, there are more studies on the detection and mechanism of multi-drug-resistant tuberculous meningitis. Hold on. Objective To investigate the difference in the dose of rifampin and the treatment time of rifampin in the treatment of tuberculosis and tuberculous meningitis. the detection method of the single, high-flux and low-cost can detect the mycobacterium tuberculosis drug-resistant mutant gene, synovitis The method of the invention compares the mycobacterium tuberculosis rifampin resistance gene mutation mechanism of the tuberculous meningitis and the mycobacterium tuberculosis mycobacterium tuberculosis drug resistance gene mutation site by the method of sequencing, and selects the drug resistance gene mutation site of the mycobacterium tuberculosis mycobacterium tuberculosis mycobacterium tuberculosis drug resistance gene mutation mechanism and the tuberculosis drug resistance database, The high-confidence resistance site was obtained. The rpoB gene was obtained through the GENE database of NCBI (Rv0667). The primers were designed according to the principle of M-ARMS-PCR. The five pairs of primers were 1037OF-1048OR, 511IF-1478OR, 55IF-1478OR, 526IF-1478OR, 531IF-1478OR, and 5 individual upstream primers were used to form 4 pairs of primers with the downstream 1478OR to form half-nested PCR and the product was large. Small:442 bp,227 bp,214 bp,18 The M-ARMS-PCR was amplified by M-ARMS-PCR and QIAxce was used. 1. Detection and analysis of the instrument. The drug resistance of these 135 strains was determined. DNA sequencing was carried out in the region. The results of drug sensitivity and sequencing showed that M-ARMS- PC Evaluation of R. Results The results of this study were compared to the distribution of rifampin-resistant Mycobacterium tuberculosis and rifampin-resistant rpoB gene. The most common site of rpoB gene mutation in tuberculous meningitis was 531 (53.97%),526 (20.63%) and 516 (6.45%), and the most common site of rpoB gene mutation in tuberculous meningitis was 531 (61.54%),526 (20.51 (%),533 (7.69%). The detection of rifampin-resistant gene rpoB based on the QIAxcel detection platform was established. -ARMS-PCR detection method. This method is capable of amplifying the rpoB gene by a single-tube 5-weight selective PCR with four codons 511,516,526,531, Wild-type gene, no mutant. M-ARMS-PCR result,131 Example samples were consistent with the DNA sequencing results. The sensitivity of the M-ARMS-PCR method was evaluated using a DNA sequencing method to be 9 The sensitivity of the method for evaluating M-ARMS-PCR was 86.5. 7% And the specificity is 89.71%. The common mutation area is the 531-533 area. The 531-bit password The detection method of M-ARMS-PCR based on QIAxcel detection platform detects rifampin resistance gene mutation and can be used for 6 hours. The method has the advantages of convenience, rapidness, accuracy, early and low cost,
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R529.3

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相關期刊論文 前2條

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本文編號：2510623