结核分枝杆菌耐药性分析及利福平和氧氟沙星耐药基因的检测
发布时间:2018-03-25 08:47
本文选题:结核分枝杆菌 切入点:耐多药结核 出处:《湖南师范大学》2015年硕士论文
【摘要】:目的:调查结核患者中结核分枝杆菌的耐药性,分析耐药结核分枝杆菌临床分离株的rpoB、gyrA和gyrB基因的突变情况,探讨一线药物利福平、二线药物氧氟沙星的耐药产生与rpoB基因、gyrA和gyrB基因突变的关系,为结核分枝杆菌(MTB)的耐药性研究、耐多药结核病(MDR-TB)和广泛耐药结核病(XDR-TB)的诊断与治疗提供新的实验依据。方法:①收集湖南省胸科医院6535例就诊的肺结核患者的标本,进行结核分枝杆菌液体培养及绝对浓度法药物敏感性实验(简称“药敏实验”,DST),检测培养菌株对链霉素(SM,S)、异烟肼(INH,H)、利福平(RFP,R)、乙胺丁醇(EMB,E)、卡那霉素(KM,K)和氧氟沙星(OFX,O)6种药物的敏感性,以结核分枝杆菌标准菌株H37Rv为对照,获得菌株对6种药物的耐药谱。②选取利福平耐药菌株,用超声波裂解法提取MTB基因组DNA,对与利福平耐药相关的rpoB基因进行扩增、测序并比对其突变情况,分析基因突变和耐药表型的关系。③选取氧氟沙星耐药菌株,采用超声波裂解法提取MTB基因组DNA,对与氧氟沙星耐药相关的gyrA和gyr B基因进行扩增、测序,分析该两个基因突变与耐药表型的相关性。结果:①结核分枝杆菌培养阳性1140例,初治患者826例,复治患者314例;耐药肺结核患者443例,其中耐药初治患者296例,耐药复治患者147例,总耐药率为38.85%(443/1140),初治患者总耐药率为35.84%(296/826),复治患者总耐药率为46.81%(147/314)。耐多药患者270例,耐多药率为23.68%(270/1140),其中同时耐利福平、异烟肼和链霉素在耐多药模式中所占比例最高,达32.22%(87/270)。而单一耐药以利福平最高,耐单药率为28.51%(325/1140)。②本地区rpoB基因突变以C531T(Ser→Leu)为主,占69.64%(39/56),其中耐多药菌株有29株(70.73%),其次是发生在RRDR-526位点上的基因突变,占16.07%。③1140株阳性标本中有140株对氧氟沙星耐药,单耐药率高达12.28%,是二线药物中耐药率最高的一种药物,而在32株氧氟沙星耐药的MTB中,所测的gyr基因突变共有25株,且均为gyrA基因突变,其突变率为78.1%(25/32)。gyrA基因突变株中,以94位点较为常见,占gyrA基因突变的60.0%(15/25),而在gyrB基因上均未发现突变位点。结论:①某院就诊的结核患者的结核分枝杆菌总耐药率和耐多药率分别为38.85%和23.68%。②rpoB基因的81bp耐药区域上以RRDR-531和RRDR-526两个位点上的突变为主要突变形式。③氧氟沙星耐药与gyr基因突变有一定的关系,gyrA基因突变是主要突变形式。
[Abstract]:Objective: to investigate the drug resistance of Mycobacterium tuberculosis in patients with tuberculosis, to analyze the mutation of rpoB gyrA and gyrB genes in clinical isolates of Mycobacterium tuberculosis, and to explore the first-line drug rifampicin. The relationship between drug resistance of the second line drug ofloxacin and mutation of rpoB gene gyrA and gyrB gene is a drug resistance study of Mycobacterium tuberculosis (Mycobacterium tuberculosis). The diagnosis and treatment of MDR-TB and XDR-TB) provided a new experimental basis for the diagnosis and treatment of MDR-TB.Methods the specimens of 6535 pulmonary tuberculosis patients in Hunan Provincial chest Hospital were collected. The susceptibility of mycobacterium tuberculosis to Streptomycin (SMC), isoniazid (INHH), rifampicin, EMBB, kanamycin (KMK) and ofloxacin (OFX) OFX was tested by liquid culture and absolute concentration assay (DST). Using Mycobacterium tuberculosis standard strain H37Rv as control, the rifampicin resistant strains were selected from the resistant spectrum of 6 kinds of drugs. The MTB genomic DNA was extracted by ultrasonic lysis method, and the rpoB genes related to rifampicin resistance were amplified. The relationship between gene mutation and drug resistance phenotype was analyzed. 3 ofloxacin resistant strains were selected. The MTB genomic DNA was extracted by ultrasonic lysis method. The gyrA and gyr B genes associated with ofloxacin resistance were amplified and sequenced. Results 1140 cases of Mycobacterium tuberculosis culture positive, 826 cases of initial treatment, 314 cases of re-treatment, 443 cases of drug-resistant pulmonary tuberculosis, including 296 cases of drug-resistant initial treatment, were analyzed. 147 patients were re-treated with drug resistance, the total drug resistance rate was 38.85% / 1140%, the total drug resistance rate of the first treatment patients was 35.84 / 296 / 826, and the total drug resistance rate of the re-treated patients was 46.81% 1477 / 3140.The multidrug resistance rate of 270 patients with multidrug resistance was 23.68,280 / 270,114040%, among which rifampicin was also resistant to rifampicin. Isoniazid and streptomycin accounted for the highest proportion of multidrug resistance patterns, reaching 32.220.87 / 2700.The single drug resistance of rifampicin was the highest, and the rate of single drug resistance was 28.51x / 1140.2 the mutation of rpoB gene in this region was C531T(Ser. 鈫扡eu) was predominant, accounting for 69. 64% of 39 / 56%, of which 29 strains were multidrug resistant strains, followed by gene mutations at the RRDR-526 locus, accounting for 140 of 16. 07% of positive specimens. The rate of single drug resistance was 12. 28%, which was the highest among the second line drugs. Among 32 strains of MTB resistant to ofloxacin, 25 gyr gene mutations were detected, all of which were gyrA gene mutations. The mutation rate was that of 78.1%(25/32).gyrA gene mutants, 94 loci were more common. 60.015 / 25% of the mutation in gyrA gene, but no mutation site was found on the gyrB gene. Conclusion the total drug resistance rate and multidrug resistance rate of Mycobacterium tuberculosis patients in one hospital are 38.85% and RRDR-531 and RRDR-526 in 81bp resistance region of 23.68%.2rpoB gene are 38.85%, respectively. The mutation at two loci was the main mutation form. 3 ofloxacin resistance was related to the mutation of gyr gene, and the mutation of gyra gene was the main mutation form.
【学位授予单位】:湖南师范大学
【学位级别】:硕士
【学位授予年份】:2015
【分类号】:R440
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