浙江省耐药结核分枝杆菌katG、rpoB、embB基因突变特点研究

目的 了解浙江省耐异烟肼、利福平、乙胺丁醇结核分枝杆菌katG、rpoB、embB基因突变特点.方法 以结核分枝杆菌标准株H37Rv为对照,应用聚合酶链反应--单链构象多态性(PCR-SSCP)技术、聚合酶链反应--直接测序(PCR-DS)技术检测100株耐药结核分枝杆菌临床分离株katG、rpoB、embB基因.结果 经PCR-DS检测katG基因突变率达54.8%,以Ser315Thr突变最常见(40.3%);在耐利福平菌株中,rpoB基因突变率达88.9%,以526、531位氨基酸改变最常见,总突变率达77.8%(63/81);在耐乙胺丁醇菌株中,embB基因突变率达60%,以Met306VaI改变最常见(28.9%).结论 PCR-SSCP技术操作简单、检测快速,可作为临床耐药检测的辅助手段;浙江省结核菌耐药基因突变位点同国内研究基本一致,各位点突变形式所占比例有自身特点.     

基因芯片方法快速检测MDR-TB及利福平和异烟肼耐药基因的研究

目的 使用基因芯片结合仪器法液体快速培养分析耐多药(multidrug resistant,MDR)结核分枝杆菌(mycobacterium tuberculosis,MTB)的耐药基因和表型特征.方法 应用聚合酶链反应(polymerase chain reaction,PCR)扩增-基因芯片杂交法检测耐多药结核分枝杆菌(multidrug resistant tuberculosis,MDR-TB)菌株中利福平(rifampin,RFP)和异烟肼(isoniazid,INH)耐药基因的突变位点及类型,平行用BD MGIT960系统检测所选菌株对RFP和INH的敏感性.结果 以MGIT960药敏结果作为参考标准,34株MDR-TB中,基因芯片检测MTB对RFP、INH药的符合率分别为85.29%和94.11%;32株RFP耐药突变株中22株为rpoB 基因531位密码子突变,29株INH耐药突变株中24株为katG基因315位密码子突变.结论 基因芯片技术可快速、有效地检出MDR-TB,可以在未获得传统细菌表型药敏结果前指导临床用药治疗.     

结核分支杆菌耐药分子机制及快速检测的研究

目的：研究结核分支杆菌异烟肼、利福平耐药的分子机制，探索快速检测结核分支杆菌异烟肼、利福平耐药性的分子生物学方法。方法：应用聚合酶链反应－单链构象多态性（PCR－SSCP）检测结核分支杆菌异烟肼、利福平耐药株与katG基因、rpoB基因突变。结果：46例株结核分支杆菌临床分离株均未发现katG基因序列的缺失，17株检测到katG基因突变，耐药株中katG基因的突变率为57％；87株结核分支杆菌利福平耐药临床分离株的PCR－SSCP结果显示，所有39株利福平敏感菌无突变检出，48株利福平耐药菌中，36株高度利福平耐药菌和7株低度利福平耐药菌检测到rpoB基因突变；另5株低度利福平耐药菌未检测到rpoB基因突变，利福平耐药株中rpoB基因的突变检出率为89．6％。结论：证实katG和rpoB基因突变分别是结核分支杆菌异烟肼和利福平耐药的主要分子机制。应用聚合酶链反应－单链构象多态性（PCR－SSCP）可快速检测结核分支杆菌异烟肼、利福平耐药性，使应用传统耐药测定方法需时1－2个月缩短到2天内即可完成，使非生长依赖性分子生物学手段应用于快速检测结核分支杆菌耐药性成为可能。     

利用GenoType~? MTBDRplus技术分析福州市结核耐药基因突变特征

目的了解福州市结核菌的耐药基因突变特征,分析突变位点和突变频率,为本地区耐多药结核病诊疗提供更多的数据。方法利用GenoType~?MTBDRplus技术对福州市近年分离的352株结核分枝杆菌进行检测,分析其rpoB、katG和inhA基因的突变特征。结果福州市352株结核菌株,总耐药率13.92%(49/352),ropB耐药基因突变率7.67%(27/352),异烟肼耐药基因突变率13.92%(49/352),耐多药结核菌(MDR-TB)发生率7.67%(27/352)。利福平耐药基因rpoB突变以S531L位点突变最常见,占55.56%(15/27)。异烟肼耐药katG基因突变占75.51%(37/49),inhA突变占24.49%(12/49)。katG突变以S315T1最常见,占59.18%(29/49);inhA基因突变以inhA C15T最常见,占22.45%(11/49)。MDR-TB以rpoB S531L-katG S315T1突变类型最常见,占40.74%(11/27)。结论福州地区结核分枝杆菌对利福平、异烟肼和MDR-TB耐药率总体水平均低于全国平均水平,耐利福平、耐异烟肼菌株和MDR-TB的基因突变存在主流突变类型,分别为rpoB S531L、katG S315T1和inhA C15T、rpoB S531L-katG S315T1,对今后福州市耐多药结核病的快速诊断和控制提供了理论依据。     

耐多药结核分枝杆菌基因突变在耐药性检测中的应用

目的:探讨结核分枝杆菌耐链霉素(SM)和利福平(RFP)的耐药分子机制,应用聚合酶链反应一单链构象多态性(PCR-SSCP)同时检测rpsL基因、rpoB基因突变在结核分枝杆菌耐SM和RFP耐药性测定中的应用价值。方法:采用PCR-SSCP技术对168株结核分枝杆菌临床分离株rpsL基因、rpoB基因突变同时进行检测。即首先用PCR方法同时扩增RFP、SM的rpoB基因和rpsL基因,然后进行SSCP分析。结果:以结核分枝杆菌H37RV为对照,82株药物敏感株的rpsL基因、rpoB基因均未见SSCP图谱异常。特异性为100%。86株同时耐SM和RFP的耐药株中,68株(79.1%)有rpsL基因图谱异常;81株(94.2%)有rpoB基因图谱异常。结论:rpoB基因突变和rpsL基因突变分别是结核分枝杆菌耐RFP和SM的主要分子机制。应用PCR-SSCP技术可同时快速检测结核分枝杆菌SM和RFP耐药性。     

PCR-SSCP快速检测耐多药结核分枝杆菌

目的:了解本地区结核病耐药基因突变情况,探讨PCR-SSCP作为新的分子药敏试验方法在临床的应用价值。方法:通过提取耐INH、RFP、SM的肺结核患者痰中结核分枝杆菌DNA,进行PCR-SSCP分析,检测结核分枝杆菌rpoB、katG、rpsL基因是否存在突变,并与传统L-J药敏实验对照。结果:30株耐多药株中,耐RPF、INH、SM基因突变阳性率为90%(27/30)、63%(19/30)、53%(16/30)。3个基因联合突变共8株(26.7%),2个基因联合突变共18株(60%),即26株(86.7%)。单基因突变共2株,2株无基因突变。结论:通过PCR-SSCP方法可检测出绝大部分耐多药结核病的耐药基因,rpoB、katG、rpsL基因突变与本地区结核杆菌对RFP、INH、SM耐药性有关。与传统L-J药敏实验对比,PCR-SSCP是一种敏感、快速的指导临床用药的先进检测方法。     

PCR-DNA序列测定法检测脊柱结核分枝杆菌耐药基因的研究

目的 研究聚合酶链反应、DNA序列测定在脊柱结核分枝杆菌耐药性临床应用的价值.方法 对25例脊柱结核病例脓液、干酪样坏死组织,采用PCR法扩增样本的耐药基因rpoB、rpsL、katG片段 以PCR-DNA序列测定法检测扩增产物,利用DNATools（5.1）软件、NCBI/BLAST公共数据库对耐药基因比较分析.结果 73份耐药基因片段, 检测出15份基因突变,其中rpsL基因突变4份,rpoB基因突变5份,katG基因突变6份,平均报告时间6d.结论 脊柱结核分枝杆菌耐药基因序列检测具有快速、灵敏和特异性高等特点,与细菌学等方法相结合,能为脊柱结核临床治疗提供有利的帮助.     

耐多药结核分枝杆菌KatG及rpoB基因变异的研究

目的建立快速检测耐多药结核分枝杆菌的分子药敏方法,掌握盐城、南通地区结核分枝杆菌KatG及rpoB基因的突变特点,了解耐药分子机制及探索痰标本结核分枝杆菌耐INH、RFP直接检测的可行性。方法对收集的47例耐INH、RFP的结核分枝杆菌临床痰分离株行PCR-自动测序法检测KatG及rpoB基因突变,以结核分枝杆菌标准株H37Rv为参比菌株,应用BACTEC460TB快速培养系统进行自动培养、药敏。结果47例耐INH、RFP分离株结核分枝杆菌KatG及rpoB基因突变阳性分别为29/47、39/47,两者同时突变率为47%。结论PCR-直接测序法敏感、特异,是可靠快速检测结核分枝杆菌KatG及rpoB基因突变,适合于临床肺结核患者MDR-TB痰标本耐药性的快速检测。     

线性探针杂交技术在广西百色地区耐药结核病检测中的应用

目的 了解广西百色地区利福平、异烟肼耐药基因的分布情况。评价线性探针杂交技术(LPA)检测耐药结核分枝杆菌的应用价值。方法 利用LPA技术与传统比例药敏试验分别检测496例结核分枝杆菌感染患者痰标本,统计出利福平、异烟肼各个耐药基因的分布情况并与传统比例药敏试验作比较。结果 LPA技术对结核病患者耐多药的检出率和传统比例药敏试验相比较差异无统计学意义(P>0.05) 。LPA技术检测耐利福平和异烟肼的灵敏度、特异度分别为97.0%、99.1%和77.8%、 99.5%,耐多药结核病的灵敏度和特异度分别为82.8%和99.8%。本地区利福平耐药基因突变频率最高的是rpoB WT8 占32.4%,其次是ropB MUT3占29.7%;异烟肼耐药基因最常见的突变是 katG WT 占46.8%,其次是katG MUT1 占43.6%。kappa一致性检验评价两种药物耐药性检测方法的kappa值分别为0.965和0.904。结论 广西百色地区结核分枝杆菌利福平的耐药基因以rpoB WT8和ropB MUT3两个位点突变为主,而结核分枝杆菌异烟肼的耐药基因则以katG WT和katG MUT1位点突变多见。LPA技术能快速、准确诊断结核分枝杆菌利福平、异烟肼的耐药性及多药耐药性,LPA技术检测结核分枝杆菌利福平和异烟肼耐药性与传统比例药敏试验具有高度的一致性,LPA检测技术可以大大地缩短检测的时限,辅助临床快速诊断出多耐药的结核菌。     

应用基因芯片技术检测结核分枝杆菌耐异烟肼基因型

目的研制一种新型DNA芯片,用于快速检测结核分枝杆菌耐异烟肼katG基因突变。方法根据结核分枝杆菌katG基因序列设计探针并制作DNA芯片,用四甲基罗丹明标记的引物扩增结核分枝杆菌katG基因突变热点的片断,与DNA芯片杂交,同时以聚合酶链反应-单链构象多态性(pdymerase chain reaction-single stranded conformation polymorphism,PCR-SSCP)和DNA测序法为对照。结果153株结核分枝杆菌临床分离株中30株异烟肼敏感株的PCR-SSCP和DNA芯片杂交结果与结核分枝杆菌标准株完全相同,123株异烟肼耐药株中有85株检测到katG基因突变,占69.1%(85/123)。其中83株为315位密码子AGC→ACC突变,2株为315位密码子AGC→AAC突变,该突变与PCR-SSCP和DNA芯片杂交结果一致;余38株未检测到突变,经测序证实其中1株PCR-SSCP有不同突变的为279位密码子GGC→GAC突变,因芯片上未点该位点的探针,所以杂交结果为阴性。结论用DNA芯片可快速、特异地检测出大多数结核分枝杆菌耐异烟肼分离株的katG基因突变,可用于临床耐药性的检测,指导临床用药。     

Molecular fingerprinting of multidrug-resistant Mycobacterium tuberculosis strains in Beirut reveals genetic diversity and father to daughter transmission

The typing of six consecutive multidrug-resistant Mycobacterium tuberculosis strains isolated from patients with tuberculosis (TB) at the American University of Beirut Medical Center, was performed by touchdown double-repetitive-element (DRE)-PCR. The isolates exhibited four distinct patterns in DRE-PCR with three isolates exhibiting unique patterns and three isolates yielded similar DNA fragment patterns (cluster pattern). Only two of the three cluster isolates exhibited identical patterns as revealed by restriction fragment length polymorphism (RFLP) targeting specific mutations in the rpoB and katG genes that confer resistance to rifampin and isoniazid, respectively. A direct epidemiological linkage for the two isolates exhibiting genotypic relatedness was also established as the isolates were recovered from a 33-year-old man and his 8-year-old daughter. The data show that transmission of multidrug-resistant M. tuberculosis strains is contributing to the emergence of drug-resistant TB in Beirut. Combining DRE-PCR with RFLP at the rpoB and katG genes could provide a powerful means for investigating the spread of multidrug-resistant M. tuberculosis strains in Lebanon.     

Molecular characteristics of rifampin and isoniazid resistant Mycobacterium tuberculosis strains from Beijing, China

Background China is one of the high burden countries of Mycobacterium tuberculosis （TB） infection globally, with high incidence and mortality. We studied the molecular characteristics of rifampin （RIF） and isoniazid （INH） resistant Mycobacterium tuberculosis strains from Beijing, China, in order to find out the genetic marker for rapid detection of specific drug resistance. Methods Forty pansusceptible and 81 resistant strains of Mycobacterium tuberculosis isolated from Beijing, China during 2002-2005 were analyzed. The modified rifampin oligonucleotide （RIFO） assay based on reverse line blot hybridization was used to detect mutations in the 81 bp hot-spot region of rpoB gene, which is associated with RIF resistance. The INH resistance associated genes, regulatory region mab-inhA （-15C/T） and structural gene katG S315T were detected by reverse line blot hybridization and PCR-restriction fragment length polymorphism （RFLP） method respectively. All the strains were typed by spoligotying and the Beijing genotype was further subdivided by NTF locus analysis. The distribution of drug resistance associated mutations in the above genes was compared in these groups. Results Sixty-five （91.5%） of 71 RIF resistant and 52 （92.9%） of 56 multidrug-resistant （MDR, i.e. resistant to at least RIF and INH） strains were found to harbor mutations in the rpoB hot-spot region. No mutation was detected in RIF sensitive strains. The specificity and sensitivity of the modified RIFO assay were 100% and 91.5%, respectively, katG315 AGC〉ACC and inhA-15C〉T mutations were found in 40 （60.6%） and 10 （15.2%） of 66 INH resistant strains, respectively; 7.6% of INH-resistant strains had mutations in both of these genes. Therefore, a combined use of both katG315 and inhA-15 identified 68.2% of INH-resistant strains. The Beijing genotype accounted for 91.7% of total strains and was further subdivided into ＂modern＂ （76.6%） and ＂ancestral＂ （23.4%） group. There is no significant difference between ＂ancestral＂ and ＂modern＂ group in prevalance of drug resistance-associated gene mutations. Conclusions The hot-spot region of rpoB gene can be used as genetic marker for detection of RIF resistant strains; a combined use of both katG315 and inhA-15 can improve the detection rate of I NH resistant strains; the Beijing genotype is prevalent in Beijing, China; the modified RIFO assay can be a practical tool for rapid detection of RIF resistant and MDR isolates in the routine diagnostic work.     

Detecting drug resistant genetic mutation among pneumoconiosis patients complicated with tuberculosis in Mycobacterium tuberculosis L-forms application of PCR-SSCP technique in Huainan mining district

Objective： To study the relationship between drug resistant genetic mutation and drug resistance in Mycobacterium tuberculosis L-form, discuss the internal relationship between drug resistances and drug-resistant related genes and explore the value of PCR- SSCP to clinical application. Methods： A total of 52 clinically isolated strains of tuberculosis L-form were collected among 97 pneumoconiosis patients complicated with tuberculosis. The gene mutations of katG, rpoB and rpsL were detected by PCR-SSCP, and the results were compared with those analyzed by traditional antimicrobial susceptibility test（AST）. Results： The gene muta- tion rates of katG, rpoB and rpsL by PCR-SSCP were respectively 57.70% （30/52）, 65.38% （32/52） and 40.38% （21/52）. The rate of reversion was 78.85%（41/52） and the result of drag-resistant genes was invariable. The results of AST showed that there were 40 （76.92%） multi-drug resistant strains in 52 clinically isolated strains. The number for three-drug resistant strain was 21 （40.38%） and that of two-drug resistant was 19（36.54%）, but only 12（23.08%） strains were one drug resistant. The rate of total drug-resistance was 100%, but there were 15 strains of allied mutation of three genes, 16 of two mutations and 6 of only one by PCR-SSCP. The coincidences were respectively 71.43%, 84.12% and 50.00%. Then there was no significant difference between the allied mutations of multi-drug resistant gene and the mutations of only one drug resistant gene （P 〉 0.05）. Conclusion： PCR-SSCP technique has a higher sensibility and specificity to detect the genes of katG, rpoB and rpsL in tuberculosis L-form among pneumoconiosis complicated with tuberculosis,and the detecting rate of two drug resistant strains and three drug resistant strains was higher. The combined application of PCR-SSCP and AST has advantages at earlier diagnosis and guidance of clinical medications.     

结核分枝杆菌耐异烟肼katG基因LiPA检测研究

目的利用LiPA技术检测结核分枝杆菌耐异烟肼katG基因,评价LiPA技术临床应用的可行性。方法应用比例法确定80株结核分枝杆菌临床分离株其耐药性,用katG基因PCR扩增产物进行DNA测序,然后用LiPA检测技术检测结核分枝杆菌katG基因的突变,对比3种方法的检测结果,评估LiPA检测技术的准确性。结果在80株结核分枝杆菌中,35株为异烟肼敏感株,45株为异烟肼耐药株。敏感株katG基因扩增阳性率为100％,耐药株为91．1％（41／45）。耐异烟肼茵株katG的缺失率为8．9％。41株扩增阳性耐药株中有26株检测到315位密码子的突变,分别为ACC（60．97％,25／41）,ATC（2．44％,1／41）,无突变（36．58％,15／41）。LiPA技术检测、药物敏感性试验和基因测序的结果基本一致。结论结核分枝杆菌katG基因突变的LiPA技术敏感性高,特异性强,可用于结核分枝杆菌的快速药物敏感性试验评价。     

Molecular mechanisms of drug resistance in Mycobacterium tuberculosis clinical isolates

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结核分枝杆菌耐药基因rpoB.katG.inhA突变快速检测方法研究

目的 通过PCR及芯片杂交技术平台,建立临床样品中结核分枝杆菌及耐药基因突变的快速诊断新方法.方法 根据结核分支杆菌标准株H37Rv序列,我们设计了覆盖rpoB基因81个碱基高变区的5个正常探针和6个可以探测特定突变类型的突变探针,制作膜芯片,并检测临床样品中结核杆菌的基突变情况,以此来判断耐药结果.结果 18个利福平培养耐药中的15个样品都在rpoB基因上检出有突变,利福平耐药突变检出率为83.0%（18/15）;25个利福平敏感样品rpoB基因上都未检出突变.20个异烟肼培养耐药的样品中有16个在katG或inhA基因上检出有突变,异烟肼耐药突变检出率为80.0%（16/20）;25个异烟肼敏感样品katG或inhA基因上都未检出突变.结论 用膜芯片检测结核分枝杆菌对利福平和异烟肼的耐药性,具有较高的特异性和敏感性,可用于临床结核杆菌耐药性检测.并具有快速、简便、敏感的特点.     

河南省耐多药结核分枝杆菌利福平耐药基因rpoB的突变特征

目的:分析河南省耐多药(MDR)结核分枝杆菌利福平耐药相关基因rpoB的突变特征。方法:收集150株MDR结核分枝杆菌,应用PCR扩增利福平耐药相关基因rpoB编码区全长并测序,以结核分枝杆菌H37Rv标准株DNA序列为参比序列,分析菌株的突变特征。结果:MDR菌株rpoB基因编码区存在突变者占97.2%(140/144),突变在利福平耐药决定区(RRDR)内者占96.5%(139/144),RRDR内和RRDR外同时存在突变者占99.3%(139/140);突变热点依次为rpoB531、rpoB526和rpoB516。发现未被记录的15个突变位点和4个突变类型,其中rpoB1284位点突变可能是多态性位点,与利福平耐药无关。结论:检测河南地区结核分枝杆菌rpoB基因RRDR内位点的突变可预测MDR结核分枝杆菌的耐药性。     

临床肺结核治疗中耐药性分析及对策

目的:了解从分离自肺结核患者的结核分枝杆菌耐药基因突变率及耐药情况,以利抗结核治疗中药物的合理选用。方法:对痰涂片阳性的新发初治和复治肺结核患者进行痰结核分枝杆菌培养,阳性菌株采用高、低两种药物浓度,四种抗结核药物耐药性测试,同时用实时PCR法对结核分枝杆菌的耐药基因和突变进行检测。结果:127例痰培养阳性菌株总耐药率为14.2%,其中初治耐药率8.1%,复治耐药率35.7%,对四种抗结核药物的耐药率依次为异烟肼8.7%,利福平2.4%,链霉素2.4%,乙胺丁醇0.8%。耐药基因检测结果,在初治组中rpoB和katG的突变率为12.1%(12/99)和10.1%(10/99);复治组中rpoB和katG的突变率为32.1%(9/28)和21.4%(6/28)。结论:分离自肺结核患者的结核分枝杆菌在初始治疗时已存在耐药性,而药物治疗有可能使其耐药性增加。结果表明抗结核治疗前及在治疗过程中对结核分枝杆菌进行耐药性及耐药基因检测对指导临床抗结核治疗很有实际意义。