用反向斑点杂交法检测结核杆菌对链霉素和乙胺丁醇的耐药性

目的建立结核分支杆菌对链霉素（SM）和乙胺丁醇（EMB）耐药基因突变的快速检测方法。方法根据结核分支杆菌标准株H37Rv序列，自行设计针对rpsL和embB基因常见耐药突变的系列寡核苷酸探针，制成膜芯片，并对64例结核分支杆菌临床株的基因突变情况进行检测。结果在34株SM耐药菌中，有25株被检出在rpsL基因分析部位发生突变，检出率为73．5％（25／34），其中23株（67．6％）为第43位密码子AAG→AGG突变，2株（5．9％）为第88位密码子AAG→AGG突变；在32株EMB耐药菌中，有19株在embB基因分析部位发生突变，检出率为59．4％（19／32），其中12株（37．5％）为ATG→GTG突变；6株（18．8％）为ATG→ATA突变；1株（3．1％）为ATG-＊CTG突变。膜芯片检出的突变与基因测序结果一致。结论用膜芯片检测结核分支杆菌对链霉素和乙胺丁醇的耐药性，具有较高的特异性和敏感性，可作为常规药敏方法的补充，用于指导开展早期、有效的临床化疗。     

PCR-DNA测序技术检测结核分支杆菌耐多药基因突变的研究

目的应用PCR—DNA测序技术快速检测同时耐利福平和异烟肼结核分支杆菌分离株rpoB、KatG基因突变，评价其在检测结核分支杆菌耐多药性方面的价值。方法47株耐利福平和异烟肼结核分支杆菌临床分离株及30株结核分支杆菌敏感分离株用PCR—DNA测序技术分别检测rpoB、KatG基因突变。结果47株耐多药结核分支杆菌分离株中，检出43株rpoB基因突变，突变检出率为91．5％（43／47）；31株KatG基因突变，突变检出率为66．0％（31／47）；rpoB和KatG基因同时突变者31株，突变检出率为66．0％（31／47）。30株结核分支杆菌敏感株检出1株KatG基因突变。结论PCR—DNA测序技术方法敏感、准确、特异，可快速检测结核分支杆菌rpoB、KatG耐药基因突变，有利于耐多药结核分支杆菌耐药性的快速检测。     

基因芯片技术在乙型肝炎病毒分型和耐药突变检测中的应用

目的通过基因芯片检测系统，快速检测临床样品中乙型肝炎病毒的基因亚型和耐药突变情况。方法用基因芯片法检测352例临床样品，对慢性乙型肝炎患者感染的HBV亚型及耐药情况进行分析。结果在352例临床病例中，共检出耐药突变病例124例，检出率为35．7％。发现3种基因亚型，其中B型222例，占63．8％；C型115例，占33．0％；D型1例，占0．3％；B、C混合感染9例，占2．6％；型别未分1例，占0．3％。结论PCR与膜芯片杂交技术可检测乙型肝炎病毒基因亚型及耐药突变类型，并具有快速、高通量、敏感的特点，适合各临床医院开展应用。     

结核分支杆菌链霉素耐药基因的杂交检测

目的 探讨耐链霉素（Sm）结核分支杆菌的编码基因rpsL和ms的扩增以及突变情况。方法 采用PCR扩增技术对23株敏感株，32株耐药株，25例临床结核病人痰标本进行rpsL、ms基因扩增；并利用寡核苷酸探针反相斑点杂交技术对23株敏感株，32株耐药株，25例临床结核病人痰标本进行rpsL、ms基因突变检测。结果 23株敏感株、32株耐药株rpsL、ms基因扩增均阳性，阳性率100％；25例临床结核病人痰标本中rpsL基因扩增阳性率为72％，ms基因扩增阳性率为56％；rpsL基因突变率依次分别为4．3％、65．6％，50％；ms基因突变率依次分别为0、12．5％、7．2％。结论 耐链霉素（Sm）菌株的基因突变率明显高于药物敏感株；PER．寡核苷酸探针反相斑点杂交技术以其简便、快速、灵敏的特性能够为临床检测结核分支杆菌对链霉素（Sm）的耐药性提供初步依据。     

结核分枝杆菌对利福平和异烟肼耐药基因突变快速检测方法的建立

目的建立结核分枝杆菌对利福平和异烟肼耐药基因突变的快速检测方法。方法根据结核分枝杆菌标准株H37Rv序列，自行设计覆盖rpoB、katG、inhA基因突变区的系列寡核苷酸探针，并检测临床样品中结核分枝杆菌的基因突变情况，以此来判断耐药结果。结果在56个利福平耐药菌株中，有50个菌株都在rpoB基因上榆出有突变，利福平耐药突变检出率为89．3％（50／56）；有30个利福平敏感菌株rpoB基因上都未检出突变。有58个异烟肼培养的耐药菌株中有47个在katG或inhA基因上检出有突变，异烟肼耐药突变检出率为81．0％（47／58）；有30个异烟肼敏感菌株katG或inhA基因上未检出突变。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，可用于临床结核分枝杆菌耐药性的检测。     

结核分支杆菌耐喹诺酮等四种耐药基因检测的临床应用和评价

目的：采用聚合酶链反应-单链构象多态性分析(PCR—SSCP)检测结核分支杆菌耐药基因(rPOB、katG、rPSL和gyrA)，评估它的临床应用价值。方法：通过Bactec-960快速培养，利用PCR-SSCP和药物敏感试验了解96例肺结核病人结核分支杆菌耐药情况，分析临床治疗效果。结果：利福平、异烟肼、链霉素和喹诺酮类耐药率分别为59．4％、52．1％、62．5％和51．0％。分离株rPOB、katG、rPSL和gyrA基因突变率分别为57．3％、45，8％、58，3％和47．9％，药敏试验联合耐药基因检测指导治疗效果满意。结论：结核分支杆菌耐药与基因突变有关，耐药基因检测指导治疗是一种新探索，与药敏试验联合应用可互相弥补，可成为临床指导用药的好方法。     

结核分支杆菌喹诺酮耐药基因的研究

[目的]了解结核分支杆菌耐喹诺酮耐药基因突变情况,建立快速分子药敏实验方法。[方法]通过PCR—SSCP和直接测序技术(PCR～DS)分析结核分支杆菌的gyrA基因突变的情况。[结果]以结核分支杆菌标准菌株H37Rv和卡介苗做对照,30株喹诺酮敏感株的gyrA基因的SSCP图谱均泳动正常,测序分析与对照株相同。45株喹诺酮耐药株中,34株(75．6％)gyrA基因SSCP图谱泳动异常;测序证实10株为90位密码子的突变,24株为94位密码子突变,符合率100％。[结论]gyrA基因突变是结核分支杆菌耐喹诺酮的主要分子机制,PCR—SSCP可能成为测定部分结核分支杆菌耐喹诺酮株的简便、快速的方法．并有望直接用于临床标本喹诺酮敏感性试验。     

快速检测结核分支杆菌对异烟肼的耐药性的临床应用

目的建立快速检测结核分支杆菌耐药基因的分子药敏方法。方法要聚合酶链反应——单链构象多态性（PCR—SSCP）分析40株结核分支杆菌KatG基因突变。其中20株为异烟肼（INH）敏感株,20株为INH耐药株,用PCR—SSCP图谱鉴定扩增产物有无突变,H37RV标准株作对照．结果所有INH敏感株SSCP带普与对照相同;19株INH耐药株中8株与对照相同,11株有不同程度的差异,INH耐存KatG基因突变或缺失的阴性率为60％。结论多数结核分支杆菌INH是由于其KatGj基因突变所致,用PCR—SSCP筛选突变株可达到快速检测结核分支杆菌INH耐药的目的。     

耐利福平结核分枝杆菌rpoB基因序列研究

目的 研究杭州地区结核分枝杆菌（Mycobacterium tuberculosis）对利福平（RFP）的耐受与rpoB基因突变的关系。方法 随机挑选了90例结核杆菌感染的病例。选择了利福平作为主要的药物进行药敏实验，PCR扩增39株耐RFP结核分支杆菌的rpoB基因片段（580bp）；测定扩增片段的序列。并与美国国立生物信息中心（www．ncbi．nlm．nih．gov／nucleotide）检索获得结核杆菌野生株（利福平敏感株）序列（登陆号：AJ749948）作对比分析。结果 结果显示PCR结核测序方法得到了39例结核杆菌耐药株。51例敏感株，与药敏实验的方法检测的结果完全一致。测定的11株临床分离的耐药株中，526位或531位有突变，其中526位有突变的有7株，占耐药测定株63．6%，531位突变的有4株。占耐药测定株36．4％。未见两位点同时突变。检测的11株敏感株，未见526位或531位有突变。结论 杭州地区结核分支杆菌耐利福平的发生与rpoB基因的526位或531位突变密切相关，与国外的报告基本相似。但526位突变占耐药测定株高达63．6％。如此高比例目前国内外未见相似报道。PCR扩增和产物测序将是临床检测结核分支杆菌耐利福平和耐多药的一种迅速、准确的方法。     

结核分支杆菌rpoB基因突变与对利福平耐药的关系

目的:研究结核分支杆菌耐利福平(RFP)分离株rpoB基因突变情况并评价英应用价值.方法:采用多聚合酶链式反应-单链构象多态性分析法(PCR-SSCP法)对60株结核分支杆菌rpoB基因进行检测.结果:以结核分支杆菌标准株H37Rv为对照,60株PCR扩增阳性的结核分支杆菌中,共有32株发生突变,总突变率为53.3%(32/60);24株药物敏感株有4株rpoB基因有突变,突变率为16.7%;36株耐药株中,28株rpoB基因有突变,突变率为77.8%.耐药菌株突变率明显高于敏感菌株的突变率(P<0.05).结论:rpoB基因突变是耐RFP结核分支杆菌耐药性的主要分子机制;应用PCR-SSCP可快速检测结核分支杆菌对RFP的耐药性.     

显色法芯片检测结核分枝杆菌利福平与异烟肼耐药基因的临床应用

目的观察离心柱法抽提、显色法芯片检测结核分枝杆菌(MTB)利福平(RIF)与异烟肼(INH)耐药基因的临床应用价值。方法离心柱法直接抽提痰液中MTBDNA,显色法芯片检测69例结核患者、30例非结核患者痰液和H37Rv标准MTB株DNArpoB、katG、inhA和ahpC4个基因片段多态性。结果42例RIF耐药组rpoB区基因总突变率为90.4%,rpoB511、513、516、526、531位点突变率分别为9.5%、9.5%、7.1%、40.4%、38.0%,有1例未检出芯片信号;46例INH耐药组katG、inhA、ahpC区域突变率分别为58.6%、19.5%、6.5%,有4例未检出芯片信号;30例非结核病人痰液芯片检测结果均为阴性,H37Rv标准株为野生型。结论离心柱法抽提、显色法芯片检测结核分枝杆菌RIF和INH耐药基因方法灵敏、特异、简便,无需特殊仪器,对指导临床用药价值较大。     

Evaluation of the CombiChip Mycobacteria Drug-Resistance detection DNA chip for identifying mutations associated with resistance to isoniazid and rifampin in Mycobacterium tuberculosis

The CombiChip Mycobacteriatrade mark Drug-Resistance Detection DNA chip, recently developed by GeneIn (Pusan, South Korea), is an oligonucleotide microchip coupled with polymerase chain reaction for the detection of mutations associated with resistance to isoniazid (INH) and rifampin (RIF). This oligonucleotide chip was compared with DNA sequencing and phenotypic drug susceptibility testing with 69 INH- and/or RIF-resistant and 27 all tested drug-susceptible Mycobacterium tuberculosis isolates. Two selected codons (the katG codon 315 and inhA15) allowed identification of 84.1% of INH-resistant isolates and 100% of RIF resistance were detected by screening for 7 codons: rpoB511, rpoB513, rpoB516, rpoB522, rpoB526, rpoB531, and rpoB533. The overall specificity of this oligonucleotide chip for detecting INH and RIF resistance were 100 and 95.3%, respectively. This level of sensitivity and specificity is concordant with that from the determination of M. tuberculosis drug resistance by DNA sequencing. This oligonucleotide chip is a rapid and reliable genotypic method capable of detecting multiple mutations associated with INH and RIF resistance simultaneously in a single microchip slide.     

结核分枝杆菌耐利福平和异烟肼相关基因快速检测的应用研究

目的 利用DNA芯片检测技术直接检测痰标本中结核分枝杆菌耐利福平(RFP)、异烟肼(INH)相关的耐药基因(rpoB、katG/inhA),评价DNA芯片检测技术临床应用的可行性.方法 对586份涂阳痰标本使用L-J培养并用终点法确定其耐药性,同时利用DNA芯片检测技术检测痰标觋本中结核分枝杆菌的rpoB、katG/inhA常见基因突变位点的突变情况,比对两种方法的检测结果,对不符合的菌株测定其相应DNA序列,评估上述试验的准确性.结果 (1)586份涂阳痰标本,其中3(+)163份、2(+)204份、1(+)217份,培养阳性584份.耐药结果显示,对INH、RFP敏感的菌株分别为361株和327株,耐药菌株分别为223株和247株,其中低浓度耐药、高浓度敏感菌株分别为93株和59株,低浓度、高浓度均耐药菌株分别为130株和188株.(2)耐药基因特异性片段扩增阳性标本367份(62.8%)、阴性217份(37.2%).对INH耐药相关基因(katG/inhA)突变检出率是28.4%,突发发生位点集中在katG315位密码子(89.8%);对RFP耐药相关基因(rpoB)突变检出率是55.9%(137/247),突变发生位点主要在rpoB 531和rpoB 526位密码子,发生率分别是68.6%和16.1%.(3)对L-J药敏结果与DNA芯片检测结果不符的菌株进行DNA序列分析,发现有漏检现象.结论 DNA芯片技术直接检测样本中结核分枝杆菌的相关耐药基因存在可行性,如直接应用于临床样本检测,关键要解决样本中DNA的提取效率、PCR的扩增效率和试验的质量控制.     

Molecular characteristics of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis isolates from the Russian Federation

OBJECTIVES: Three Mycobacterium tuberculosis genetic loci--rpoB and katG genes and the fabG1(mabA)-inhA operon promoter region--were studied to reveal the mutations associated with rifampicin and isoniazid resistance. METHODS: Four hundred and twelve isolates of M. tuberculosis from different regions of the Russian Federation were collected during 1997-2005. A matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based minisequencing method was used for the detection of mutations. RESULTS: Thirteen different variants of single mutations in codons 533, 531, 526, 516, 513 and 511 of the rifampicin resistance-determining region of the rpoB gene as well as the TTG insertion in the 514a position were found among the rifampicin-resistant isolates. Single nucleotide substitutions in codons 531, 526 and 516 (64.8%, 10.3% and 7.7%, respectively) were the most prevalent mutations. Codon 526 was shown to be the most variable of all. No mutations were detected in rpoB genes for 29 (10.7%) of the rifampicin-resistant isolates. 76.9% of the isoniazid-resistant isolates carried single mutations in codon 315 of the katG gene. For another 12.9% of them, double mutations in the katG gene and the fabG1(mabA)-inhA promoter region were revealed. No mutations were detected in 8.2% of the isoniazid-resistant isolates. CONCLUSIONS: Molecular analysis of the loci of rpoB and katG genes and the inhA promoter region of 412 M. tuberculosis clinical isolates from various parts of the Russian Federation was carried out. The new MALDI-TOF MS-based method may be used for rapid and accurate monitoring of the spread of drug resistance.     

应用多重PCR-单链构象多态性分析同时检测耐异烟肼和利福平的结核分枝杆菌

目的 探讨应用多重PCR-单链构象多态性分析(multiplexpulymerase chain reaction-single strand conformation polymorphism,multi-PCR-SSCP)方法快速、特异地同时快速检测结核分枝杆菌对异烟肼和利福平耐药性的效能.方法 根据结核分枝杆菌的inhA序列、katG序列、rpoB序列,分别设计出3对特异性寡聚核苷酸引物.采用multi-PCR-SSCP技术,一次性检出耐异烟肼和利福平的结核分枝杆菌.新方法的有效性通过116株临床分离株(70株耐异烟肼,66株耐利福平)的验证.结果 名 Multi-PCR-SSCP方法检测临床分离株基因突变的有效性,以细菌培养和药敏试验结果为金标准.116株临床分离株和H37Rv标准株中除了4株katG缺失突变,其余菌株3个基因katG、inhA和rpoB在单基因PCR中都扩增成功.与H37Rv标准株相比,46株katG基因突变,14株inhA基因突变,58株rpoB基因突变.38株katG和rpoB,4株inhA和rpoB,4株inhA和katG同时突变,还有2株3个基因都有突变.multi-PCR-SSCP对于耐异烟肼和利福平的结核分枝杆菌检出的敏感度分别为80%、82%,特异度分别为100%和92%.结论 multi-PCR-SSCP方法敏感、特异,能同时快速有效地检测耐多药结核分枝杆菌,有望成为临床指导用药的好方法,为深入研究耐药基凶检测奠定了良好的基础.     

Improved real-time PCR for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis clinical isolates

In this study we designed two pairs of probes for the detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis with real-time PCR procedures. One pair of probes spans the region between codon 510 and 528 of the rpoB gene, and the other one screens for mutation at the regulatory region of the inhA gene. We have evaluated these probes in combination with two other pairs of probes previously described to detect mutations in 20 susceptible and 53 unique resistant M. tuberculosis clinical isolates. We were able to detect nine different mutations affecting five codons of the rpoB gene, two different mutations at codon 315 of the katG gene and a nucleotide substitution (C209T) in the regulatory region of the inhA gene within two hours turnaround.     

Molecular Characterization of Multidrug-Resistant Isolates of Mycobacterium tuberculosis from Patients in North India

The World Health Organization has identified India as a major hot-spot region for Mycobacterium tuberculosis infection. We have characterized the sequences of the loci associated with multidrug resistance in 126 clinical isolates of M. tuberculosis from India to identify the respective mutations. The loci selected were rpoB (rifampin), katG and the ribosomal binding site of inhA (isoniazid), gyrA and gyrB (ofloxacin), and rpsL and rrs (streptomycin). We found known as well as novel mutations at these loci. Few of the mutations at the rpoB locus could be correlated with the drug resistance levels exhibited by the M. tuberculosis isolates and occurred with frequencies different from those reported earlier. Missense mutations at codons 526 to 531 seemed to be crucial in conferring a high degree of resistance to rifampin. We identified a common Arg463Leu substitution in the katG locus and certain novel insertions and deletions. Mutations were also mapped in the ribosomal binding site of the inhA gene. A Ser95Thr substitution in the gyrA locus was the most common mutation observed in ofloxacin-resistant isolates. A few isolates showed other mutations in this locus. Seven streptomycin-resistant isolates had a silent mutation at the lysine residue at position 121. While certain mutations are widely present, pointing to the magnitude of the polymorphisms at these loci, others are not common, suggesting diversity in the multidrug-resistant M. tuberculosis strains prevalent in this region. Our results additionally have implications for the development of methods for multidrug resistance detection and are also relevant in the shaping of future clinical treatment regimens and drug design strategies.     

Molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolated in Nepal

Despite the fact that Nepal is one of the first countries globally to introduce multidrug-resistant tuberculosis (MDR-TB) case management, the number of MDR-TB cases is continuing to rise in Nepal. Rapid molecular tests applicable in this setting to identify resistant organisms would be an effective tool in reversing this trend. To develop such tools, information about the frequency and distribution of mutations that are associated with phenotypic drug resistance in Mycobacterium tuberculosis is required. In the present study, we investigated the prevalence of mutations in rpoB and katG genes and the inhA promoter region in 158 M. tuberculosis isolates (109 phenotypically MDR and 49 non-MDR isolates collected in Nepal) by DNA sequencing. Mutations affecting the 81-bp rifampin (RIF) resistance-determining region (RRDR) of rpoB were identified in 106 of 109 (97.3%) RIF-resistant isolates. Codons 531, 526, and 516 were the most commonly affected, at percentages of 58.7, 15.6, and 15.6%, respectively. Of 113 isoniazid (INH)-resistant isolates, 99 (87.6%) had mutations in the katG gene, with Ser315Thr being the most prevalent (81.4%) substitution. Mutations in the inhA promoter region were detected in 14 (12.4%) INH-resistant isolates. The results from this study provide an overview of the current situation of RIF and INH resistance in M. tuberculosis in Nepal and can serve as a basis for developing or improving rapid molecular tests to monitor drug-resistant strains in this country.     

DNA芯片快速检测耐利福平结核分枝杆菌rpoB基因突变

目的 开发快速检测耐利福平结核分枝杆菌(结核菌)rpoB基因突变的DNA芯片。方法 根据结核菌rpoB基因序列设计探针并制作基因芯片，从临床样品中分离出结核菌的基因组DNA，PCR扩增含有rpoB基因突变位点的特异DNA片段，荧光标记后与芯片上含有的检测特异突变位点的寡核苷酸探针进行杂交，同时与DNA直接测序法测定序列比较。结果 35株耐利福平结核菌中有91．4％(32／35)用直接测序法检测出存在rpoB基因突变，DNA芯片的检测效率为71．4％(25／35)。结论 用DNA芯片检测结核菌对利福平的耐药性具有较高的特异性和敏感性，可用于临床结核菌耐药性检测。     

耐多药结核分枝杆菌耐药相关基因突变特征分析

目的了解中国耐多药结核分枝杆菌耐药相关基因的分子特征。方法对138株耐多药结核分枝杆菌和45株敏感菌的耐药相关基因inhA、katG和oxyR-ahpC间隔区(异烟肼)、rpob(利福平)、gyrA(氧氟沙星)和rrs(卡那霉素)进行序列测定,分析其基因突变特点。结果 138株耐多药结核分枝杆菌中,14.4%的菌株inhA基因发生突变,72.5%菌株的katG基因发生突变,15.9%菌株的oxyR-ahpC基因发生突变,同时考虑这3种基因,异烟肼耐药相关基因突变检出率可达90.6%;94.2%菌株的rpoB基因发生突变,74.5%菌株的gyrA基因发生突变,61.1%菌株的rrs基因发生突变,主要的突变位点为katG315(66.7%),inhA-15(9.4%),oxyR-ahpC-10(5.1%),rpoB516(13.8%),526(26.1%)和531(49.3%),gyrA90(21.6%)和94(51%),rrs1401(61.1%)。结论我国耐多药结核菌异烟肼、利福平、氧氟沙星和卡那霉素耐药相关基因最常见突变为katG315、inhA-15,rpoB531、526和516,gyrA94和90,rrs1401。