华南地区380株结核分枝杆菌异烟肼耐药基因突变情况分析

目的探讨华南地区异烟肼耐药结核分枝杆菌临床分离株katG、mabA-inhA、oxyR-ahpC、kasA和ndh基因的突变特点。方法在广州地区分枝杆菌菌株库中, 选择异烟肼耐药性测定结果明确的380株来自华南地区结核病患者的结核分枝杆菌临床分离株, 其中异烟肼耐药株236株和异烟肼敏感株144株, 对380株结核分枝杆菌临床分离株的5种异烟肼耐药基因片段进行序列测定分析。描述性分析各基因突变出现的频率。结果在INH耐药株和敏感株中分别发现26个位点29种突变类型、16个位点17种突变类型, 均以katG基因315AGC(S)→ACC(T)、mabA-inhA基因-15T→C最为常见, 发生率分别为48.73%(115/236)、17.37%(41/236)和9.72%(14/144)、9.72%(14/144);在katG、mabA-inhA、oxyR-ahpC、kasA和ndh中分别发现3个位点5种突变、5个位点5种突变、11个位点13种突变、6个位点6种突变和11个位点11种突变, 在INH耐药株和敏感株中5个基因突变的发生率分别为52.12%(123/236)、22.46%(5...     

Molecular analysis of isoniazid-resistant clinical isolates of Mycobacterium tuberculosis from India

The presence of mutations in specific regions of katG, inhA, oxyR-ahpC and kasA associated with isoniazid (INH)-resistant clinical isolates of Mycobacterium tuberculosis from India were analysed by DNA sequencing. Point mutations in the katG gene at codon 315 and a mutation at codon 138 were detected in 64.3% (45/70) and 4% (1/25) of isolates, respectively. Polymorphisms at codon 463 of the katG gene were found both in resistant and sensitive isolates. Mutation at the inhA and oxyR-ahpC promoter regions occurred in 11.4% (8/70) and 35.0% (14/40) of the isolates, respectively. No mutation was found to occur in kasA and inhA structural gene regions. Of the 70 resistant isolates studied, 55 (78.6%) showed mutation in the regions sequenced. This is the first comprehensive molecular analysis of INH resistance in India, which suggests that point mutation rather than deletion and insertion is the major cause of INH resistance.     

基因芯片技术诊断耐多药结核病的临床应用研究

摘要： 目的 探讨基因芯片法检测耐多药结核分枝杆菌临床分离株耐药相关基因 rpoB、katG 和 inhA 突变的特点, 评估其临床应用价值。 方法 选取 2013—2014 年石家庄地区耐多药结核分枝杆菌临床分离株 76 株, 采用基因芯片法检测 rpoB、katG 及 inhA 基因耐药突变位点及频率, 以比例法药敏结果为金标准, 评价基因芯片法检测菌株耐药的准确度、灵敏度和特异度, Kappa 检验评价基因芯片法和比例法药敏结果的一致性。 结果 在 76 株石家庄地区耐多药结核分枝杆菌临床分离株中, 69 株检测到 katG/inhA 基因发生单一或联合突变, 其中 katG 基因优势突变位点为 315, 占 89.9%（62/69）, 5.8%（4/69）存在 inhA -15（C→T）突变, 4.3%（3/69）发生 katG 315 位点和 inhA 启动子区域的联合突变。 73 株检测到 rpoB 基因发生突变, 优势突变位点为 531, 占 64.4%（47/73）; 其次是 526, 突变率为 15.1%（11/73）; 12.3%（9/73）发生 516 位点突变; 1.4%（1/73）发生 513 位点突变; 1.4%（1/73）发生 533 位点突变; 另有 5.5%（4/73）发生多位点的联合突变。 以比例法药敏结果为金标准, 基因芯片法检测利福平耐药的敏感度为 96.1%（73/76）, 特异度为 100.0%（50/50）, 总准确度为 97.6%（123/126）; 检测异烟肼耐药的敏感度为 90.8%（69/76）, 特异度为100.0%（50/50）, 总准确度为 94.4%（119/126）; 基因芯片法检测耐多药结核的敏感度为 86.8%（66/76）, 特异度为100.0%（50/50）, 总准确度为 92.1%（116/126）。 结论 本地区耐多药结核分枝杆菌耐药相关基因优势突变类型为katG 基因 315 位点和rpoB 基因 531 位点, 基因芯片法可快速、有效诊断耐多药结核病。     

结核分枝杆菌异烟肼耐药基因katG点突变的快速检测

目的：探讨快速检测结核分枝杆菌异烟肼（INH）耐药基因型的分子药敏方法。方法：用聚合酶链反应－单链构象多态性（PCR－SSCP）检测了30株INH敏感菌株及30株耐药株的katG基因，随后用SSCP方法鉴定扩增产物有无突变，以结核分枝杆菌H37Rv作对照。结果：所有INH敏感株均观察到katG基因扩增产物，30株INH耐药株中28株观察到katG基因扩增产物。以H37Rv标准株为对照，30株敏感株SSCP带谱与对照相同；28株INH耐药株中13株与对照相同，15株有不同程度的差异。与药敏试验比较，PCR－SSCP检测INH耐药结核菌的敏感性为57%，特异性为100％。结论：多数结核分枝杆菌耐INH是由于katG基因突变所致，用PCR－SSCP筛选突变株可达到快速检测结核分枝杆菌INH耐药基因型的目的。     

结核分枝杆菌katG和inhA基因突变的研究

为了解结核分枝杆菌耐异烟肼（ＩＮＨ）分离株ｋａｔＧ基因和ｉｎｈＡ调节序列的突变情况，探讨其在ＩＮＨ耐药性中的可能作用。应用聚合酶链反应（ＰＣＲ）和ＰＣＲ－单链构象多态性（ＳＳＣＰ）方法对６２株结核分枝杆菌临床分离株的ｋａｔＧ基因和ｉｎｈＡ调节序列进行分析。ＰＣＲ分析结果显示：ｋａｔＧ基因和ｉｎｈＡ调节序列的敏感性为１～１０ｐｇＤＮＡ；其引物ＰＣＲ扩增都具有较高的特异性。以Ｈ３７Ｒｖ标准株为对照，１３株敏感株中，１２株ｋａｔＧ基因扩增产物ＳＳＣＰ泳动正常，１株ｋａｔＧ异常；２４株耐非ＩＮＨ抗结核药物株中，８株ｋａｔＧＳＳＣＰ异常；上述２７株的ｉｎｈＡ调节序列ＳＳＣＰ均泳动正常。２５株耐ＩＮＨ株中，３株ｋａｔＧ基因ＰＣＲ扩增阴性，２２株ｋａｔＧ扩增阳性，其中１６株ＳＳＣＰ泳动异常；４株ｉｎｈＡ调节序列ＳＳＣＰ泳动异常，其ｋａｔＧ基因ＳＳＣＰ也异常。结果表明，某些结核分枝杆菌ＩＮＨ耐药性可能是由于其ｋａｔＧ基因完全缺失、ｋａｔＧ基因或ｉｎｈＡ调节序列突变所致，但某些菌株也可能与其无关，是其它耐药基因所致或存在多种机制，尚需进一步探讨。     

铜绿假单胞菌临床分离株gyrA基因突变与喹诺酮类药物耐药关系研究

目的:研究铜绿假单胞菌临床分离株gyrA基因突变与喹诺酮类药物耐药关系,并对聚合酶联反应(PCR)-限制性片段长度多态性(RFLP)-DNA单链构象多态性(SSCP)分析铜绿假单胞菌临床分离株gyrA基因突变的可行性进行评估。方法:以铜绿假单胞菌临床分离株gyrA基因序列为靶序列,用PCR、PCR-RFLP、PCR-SSCP、DNA测序等方法对铜绿假单胞菌ATCC27853及16株临床分离株gyrA基因突变进行对比研究。结果:在8株耐环丙沙星铜绿假单胞菌中,有6株gyrA基因的83位表现出单点突变,其突变方式全为ACC→ATC,导致氨基酸苏氨酸→异亮氨酸的改变;gyrA基因的PCR扩增产物SacⅡ酶切片段与测序结果一致;SSCP分析结果显示,16株细菌中仅2株gyrA带型与ATCC27853相同,其它菌株gyrA带型与ATCC27853均不同。结论:临床分离的铜绿假单胞菌对喹诺酮类药物耐药的分子机制主要表现为gyrA基因83位氨基酸密码子突变,应用PCR-RFLP-SSCP系统可快速、准确地检测耐喹诺酮类药物的铜绿假单胞菌gyrA中碱基的变异。     

用分子生物学技术快速检测耐多药结核分枝杆菌

目的：用PCR—SSCP技术检测耐INH、RFP、SM结核分枝杆菌分离株KatG、rpoB、rpsL基因突变，评价其在快速检测结核分枝杆菌耐药性方面的临床价值。方法：以结核分枝杆菌H；vRv为对照，20株耐多药菌株和20株敏感株，分别用PCR—SSCP方法检测KatG、rpoB、rpsL基因突变，并将SSCP结果与药敏试验结果作对照分析。结果：PCR—SSCP方法检测20株敏感株SSCP带语均与H37RV标准株相同，而20株耐多药结核分枝杆菌KatG、rpoB、rpsL基因突变的阳性率分别为70％，100％和75％。发现20株耐多药菌株中有11株(55％)共INH、RFP、SM3种耐药遗传标志均发生突变，7株(35％)有2种耐药遗传标志突变，2株(10％)只有RFP耐药标志突变。结论：PCR—SSCP方法敏感、特异，可快速检测结核分枝杆菌KatG、rpoB、rpsL耐药基因突变，有利于耐多药结核分枝杆菌耐药性的快速检测。     

The occurrence of rare rpoB mutations in rifampicin-resistant clinical Mycobacterium tuberculosis isolates from Kuwait

Mutations associated with rifampicin (RIF) resistance in two regions of the rpoB gene were studied by line probe (INNO-LiPA Rif. TB) assay (LiPA) and/or DNA sequencing in 32 RIF-resistant and 21 RIF-susceptible Mycobacterium tuberculosis strains isolated from 53 tuberculosis patients in Kuwait. The LiPA identified all susceptible strains as RIF sensitive, and the DNA sequences of the 81bp rifampicin resistance-determining region (RRDR) and the N-terminal region of the rpoB gene from five isolates were identical to the wild-type sequences from M. tuberculosis H(37)Rv. The LiPA identified 24 of 32 (75%) phenotypically documented RIF-resistant M. tuberculosis isolates as RIF resistant, with specific detection of mutation in 19 isolates, whilst 8 strains were identified as RIF susceptible. DNA sequencing of the RRDR confirmed the results for the 19 RIF-resistant isolates and identified precise mutations in five isolates for which specific base changes could not be determined by LiPA, as well as identifying a mutation (insertion 514TTC) in three isolates and no mutation in five of the eight isolates that were detected as RIF susceptible by LiPA. Two of the latter five isolates contained a V146F mutation in the N-terminal region of the rpoB gene and were recovered from patients of Middle Eastern origin. These analyses identified 11 different mutations in two regions of the rpoB gene. The majority of the isolates carrying identical or no mutations in the rpoB gene exhibited unique DNA banding patterns in fingerprinting studies. The occurrence of rare mutations in the rpoB gene, namely insertion 514TTC in 3 (9%) and V146F in 2 (6%) of 32 RIF-resistant M. tuberculosis isolates from Kuwait, is the highest reported so far.     

广东地区结核分枝杆菌利福平耐药与基因rpoB突变关系分析

目的 为了解广东地区结核分枝杆菌rpoB基因突变特点,并探讨其与利福平耐药之间的关系.方法 本研究根据结核病诊断细菌学检验规程从临床结核病疑似患者痰液中分离鉴定结核分枝杆菌,采用绝对浓度法对其进行药敏试验,并用PCR-DNA直接测序法对结核分枝杆菌rpoB基因进行序列分析.结果 结果分离得到利福平耐药株27株,其中低浓度耐药8株,高浓度耐药19株.经测序分析,所有利福平敏感株rpoB基因均未发生氨基酸突变 利福平耐药株rpoB基因的突变率为81.5%（22/27）,突变均发生在利福平耐药决定区内,突变位点为526、531及533,以Ser531Leu突变最为常见（55.6%,15/27）,并且高浓度耐药株的突变率明显高于低浓度耐药株.结论 结果表明广东省结核分枝杆菌rpoB基因突变是结核分枝杆菌利福平耐药性产生的主要分子机制,突变位点同国内外研究基本一致,但各位点突变形式所占比例有自身特点.     

应用PCR—直接测序法测定结核分支杆菌rpsL基因突变的研究

目的：了解我国结核分支杆菌耐链霉素（Ｓｔｒｅｐｔｏｍｙｃｉｎ,ＳＭ）分离株ｒｐｓＬ基因突变情况,建立快速检测结核分支杆菌耐药基因型的分子药敏试验方法。方法：通过聚合酶链反应（ＰｏｌｙｍｅｒａｓｅＣｈａｉｎＲｅａｃｔｉｏｎ,ＰＣＲ）－单链构象多态性（Ｓｉｎｇｌｅ－ｓｔｒａｎｄｅｄＣｏｎｆｏｒｍａｔｉｏｎＰｏｌｙｍｏｒｐｈｉｓｍ,ＳＳＣＰ）、ＰＣＲ－限制性片段长度多态性（ＲｅｓｔｒｉｃｔｉｏｎＦｒａｇｍｅｎｔＬｅｎｇｔｈＰｏｌｙｍｏｒｐｈｉｓｍ,ＲＦＬＰ）和ＰＣＲ－直接测序法（ＤｉｒｅｃｔＳｅｑｕｅｎｃｉｎｇ,ＤＳ）分析３８株结核分支杆菌耐ＳＭ分离株的ｒｐｓＬ基因。结果３８株耐ＳＭ分离株中,２５株ＳＳＣＰ异常、不被ＭｂｏⅡ消化、ＤＳ分析４３位密码子ＡＡＧ→ＡＧＧ突变;１株ＳＳＣＰ异常、可被ＭｂｏⅡ消化、ＤＳ分析３３位密码子ＧＴＡ→ＡＴＡ突变;１２株ＳＳＣＰ正常、可被ＭｂｏⅡ消化、ＤＳ分析未见异常;未发现８８位密码子突变。结论：大多数结核分支杆菌耐ＳＭ是由于其ｒｐｓＬ基因４３位密码子突变所致,采用ＰＣＲ－ＳＳＣＰ、ＰＣＲ－ＲＦＬＰ和ＰＣＲ－ＤＳ方法可快速测定部分结核分支杆菌ＳＭ耐药基因型。     

宁波地区结核分枝杆菌基因突变位点确证及与异烟肼、利福平耐药关系研究

【摘要】： 目的 阐明宁波地区初治肺结核患者结核分枝杆菌rpoB、katG、inhA基因突变特征,深入分析基因突变与异烟肼（INH）、利福平(RFP)耐药关系,为临床抗结核治疗提供科学依据。方法 采用1%比例法对889例结核分枝杆菌临床分离株进行药敏检测,对其中的耐药菌株提取DNA,PCR扩增,对耐药菌株katG、inhA、rpoB基因测序,分析三种基因特征及基因突变与单耐药、耐多药关联性。结果 889例结核分枝杆菌临床分离株中,发现39例耐药结核分枝杆菌,其中单耐药24例,单耐药率2.70%,15例耐多药,耐多药率1.69%。39例耐药菌株中,有14例耐多药菌株发生rpoB、katG基因错义突变,突变率93.33%,3例发生inhA基因错义突变,突变率20.00%,rpoB基因450位点（ser450leu）突变率53.33%,katG基因315位点（ser315thr）突变率86.67%,463位点（arg463leu）突变率93.33%。3例单耐利福平菌株rpoB基因在463位点（arg463leu）全部发生错义突变。21例单耐异烟肼菌株中有19例发生katG基因错义突变,突变率90.47%,315位点（ser315thr）突变率63.16%,463位点（arg463leu）突变率78.95%;9例发生inhA基因错义突变,突变率42.86%,145位点（val145gly）突变率77.77%;5例发生rpoB基因错义突变,突变率23.81%,位点较分散。另外,39例耐药菌株中,有10例发生rpoB、katG、inhA多基因联合突变,突变率25.64%。结论 rpoB、katG、inhA基因突变与初治肺结核患者耐药密切相关,上述三种基因在450（ser450leu）、315（ser315thr）、463（arg463leu）、145（val145gly）等位点突变是本地区结核分枝杆菌对异烟肼、利福平耐药的重要机制之一。     

Detection of novel coupled mutations in the katG gene (His276Met, Gln295His and Ser315Thr) in a multidrug-resistant Mycobacterium tuberculosis strain from Chennai, India, and insight into the molecular mechanism of isoniazid resistance using structural bioinformatics approaches

This study reports on the structural basis of drug resistance targeting the katG gene in a multidrug-resistant Mycobacterium tuberculosis (MDR-TB) strain with two novel mutations (His276Met and Gln295His) in addition to the most commonly reported mutation (Ser315Thr). A structural bioinformatics approach was used to predict the structure of the mutant KatG enzyme (MT). Subsequent molecular dynamics and docking studies were performed to explain the mechanism of isoniazid (INH) resistance. The results show significant conformational changes in the structure of MT leading to a change in INH binding residues at the active site, with a significant increase in the inhibition constant (Ki) of 5.67 μm in the mutant KatG-isoniazid complex (MT-INH) compared with the wild-type KatG-isoniazid complex (WT-INH). In the case of molecular dynamics studies, root mean square deviation (RMSD) analysis of the protein backbone in simulated biological conditions revealed an unstable trajectory with higher deviations in MT throughout the simulation process (1 ns). Moreover, root mean square fluctuation (RMSF) analysis revealed an overall increase in residual fluctuations in MT compared with the wild-type KatG enzyme (WT), whilst the INH binding residues of MT showed a decreased fluctuation that can be observed as peak deviations. Hence, the present study suggests that His276Met, Gln295His and Ser315Thr mutations targeting the katG gene result in decreased stability and flexibility of the protein at INH binding residues leading to impaired enzyme function.     

结核病临床分离株及痰标本中embB基因型的快速测定

目的建立快速测定结核病耐乙胺丁醇(EMB)分离株和痰标本中结核分枝杆菌EMB耐药基因型的方法，以期为患者提供及时、有效地化验结果。方法应用16S rDNA聚合酶链反应(PCR)-单链构象多态性(SSCP)和PCR-限制性片段长度多态性(RFLP)对11株耐EMB分离株和46例结核病痰标本进行分子菌种鉴定和embB基因突变的部位与性质分析。结果以结核分枝杆菌H37Rv标准株为对照，11株耐EMB分离株和46例临床痰标本经16S rDNA PCR-SSCP分析电泳图谱均与结核分枝杆菌标准株相同；限制性内切酶NlaⅢ消化embB基因扩增产物显示，11株耐EMB分离株中4株(36．4％)不被NlaⅡ消化；46例结核病痰标本中10例(21．7％)不被NlaⅡ消化。结论部分结核分枝杆菌耐EMB是由于embB基因突变所致。采用PCR-SSCP和PCR-RFLP方法可直接快速测定结核病耐EMB分离株和痰标本中结核分枝杆菌耐EMB基因型。     

耐氟喹诺酮类铜绿假单胞菌gyrA及parC基因突变研究

目的　研究临床分离的耐氟喹诺酮类铜绿假单胞菌gyrA及parC基因突变情况。方法　测定临床分离的 5 5株铜绿假单胞菌MIC值 ,从中筛选出 1株敏感菌和 8株耐药菌 ,以标准敏感菌株ATCC2 785 3作为质控菌株。用聚合酶链反应 (PCR)扩增gyrA及parC基因的喹诺酮耐药决定区 (QR DR) ,扩增产物片段长度分别为 35 1bp、397bp。用限制性内切酶SacⅡ消化gyrAPCR产物 ,同时对上述 10株菌的gyrA及parC基因的喹诺酮决定区 (QRDR)进行PCR DNA直接测序分析。结果　有 8株耐菌株的gyrA基因在 83位 (ACC→ATC)有突变 ,导致氨基酸Thr→Ile的改变 ;有 3株高度耐药菌gyrA基因同时在 87位 (GAC→GGC)有突变 ,导致氨基酸Asp→Gly的改变 ;有 4株耐药菌株的parC基因在 87位有TCG→TTG突变 ,导致氨基酸由Ser→Leu的改变。同时具gy rA和parC突变MIC值是仅具gyrA突变菌株MIC值的 2～ 16倍。未发现parC突变单独存在。另外 ,有 6株耐药菌gyrA的 132位有CAC→CAT的突变 ;所有耐药菌株parC基因 115位有GCT→GCG的突变 ,该突变未引起氨基酸的改变。结论　gyrA83、87位突变及parC基因 87位突变都可引起铜绿假单胞菌对氟喹诺酮类药物产生耐药 ,但以gyrA基因 83位突变为主 ,合并gyrA基因 87位及parC基因 87位突变可增加耐药程度。     

PCR-SSCP技术直接快速检测痰标本中结核分支杆菌rpoB,katG,ahpC基因突变的研究

目的：应用PCR－SSCP技术直接快速检测痰标本中结核分支杆菌rpoB,katG及ahpC基因突变,评价此方法用于结核分杆菌利福平（RFP）及异烟肼（INH）耐药性试验的意义。方法：以结核分支杆菌H37Rv为对照,30例耐RFP（单耐或多耐）、21例耐INH（单耐和多耐）的结核分枝杆菌临床分离株及10例敏感菌株;39例菌阳肺结核患者及30例非结核 性肺部疾病患者痰标本,采用PCR－SS－CP技术检测痰标本和菌株中的结核杆菌rpoB,katG,ahpC基因突变,并与药敏试验对照。结果：PCR－SSCP检测痰标本中结核分支杆菌rpoB,katG及ahpC基因突变的阳性率分别为79％（31／39）、46％（18／39）及5％（2／39）.结论：PCR－SSCP可望成为直接检测临床痰标本中结核分枝杆菌耐利福平及耐异烟肼的快速方法。     

结核分支杆菌耐药基因检测结果分析

目的 分析肺结核患者结核分支杆菌获得性耐药情况,比较两种耐药性检测方法的检测效果.方法 用药敏试验(绝对浓度法)检测结核分支杆菌株对利福平(RFP)、异烟肼(INH)、链霉素(SM)、吡嗪酰胺(PZA)和乙胺丁醇(EMB)的耐药情况,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rpoB、katG、rpsL、pncA和embB基因突变.结果:400例肺结核耐药患者常规药敏试验耐药316例(79.8%);耐药基因检测耐药株288株,突变率72.0%.RFP耐药例数和耐药率明显高于SM、PZA和EMB(耐药例数:F=2.45,2.56,2.69,P＜0.05;耐药率:F=2.55,2.66,2.79,P＜0.05),rpoB突变株和突变率明显高于katG、rpsL、pncA和embB(突变株:F=2.28,2.46,3.19,3.33,P＜0.05～0.01;突变率:F=2.36,2.61,3.25,3.45,P＜0.05～0.01),高浓度药物耐药菌株的突变株和突变率显著高于低浓度药物耐药菌株,随着不规律用药时间的延长,耐药率和突变率均逐渐上升(耐药率:F=2.77,2.88,P＜0.05;突变率:F=2.72,2.85,P＜0.05).结论 PCR-SSCP是一种快速检测结核杆菌rpoB、katG、rpsL、pncA和embB基因突变敏感、特异的方法.     

遗传性高铁血红蛋白一家系的基因诊断

目的 检测Ⅰ型遗传性高铁血红蛋白血症(HM)先证者(L72P,长乐)家系成员的细胞色素b5还原酶(b5R)基因的突变。方法 PCR方法扩增正常人和HM先证者之兄、父、母的b5R基因组DNA片段(含第3和第4外显子);用限制性内切酶Apa Ⅰ分析扩增产物。结果PCR扩增产物用Apa Ⅰ酶切后,正常人为649和471 bp;先证者之兄为649、440和31bp,表明存在b5R基因第72密码子纯合子突变:其父、母均为649、471、440和31bp,表明存在b5R基因第72密码子杂合子突变。结论PCR结合Apa Ⅰ内切酶分析可用于b5R基因第72密码子突变的检测;PCR-RFLP方法是检测人群中b5R基因杂合子突变的简捷有效的方法。     

Human CYP1A1 (cytochrome P(1)450) gene: lack of association between the Msp I restriction fragment length polymorphism and incidence of lung cancer in a Norwegian population.

In this study of 221 lung cancer patients and 212 controls, no association between a Msp I polymorphism in the CYP1A1 gene and an increased risk of lung cancer was found. Histological type, smoking habits and family history were also examined. No associations between the Msp I restriction fragment length polymorphism in the CYP1A1 gene and any of these parameters were found. These results are in contrast to a previous report by a Japanese group (Kawajiri et al., 1990) who found an association between the less common allele and an increased susceptibility to lung cancer in their population. The frequency of the less common Msp I 1.9 kb fragment allele (C2) appears to be three times greater in the Japanese population than in the Norwegian population and a Caucasian population of North America. It is possible that in the Asian population this Msp I polymorphism is in linkage disequilibrium with another mutation important for CYP1A1 gene expression, whereas in the Caucasian population these mutations are in equilibrium.     

耐多药结核分枝杆菌三种基因的快速检测

目的 :探讨耐多药结核分枝杆菌耐药基因突变与耐药性的关系。方法 :采用 PCR和 PCR- DS技术对 5 7例耐多药结核临床分离株进行 kat G、rpo B和 emb B基因检测和序列分析。结果 :耐 INH(kat G)、RFP(rpo B)、EMB(emb B)基因突变率分别为 6 3.8%、90 .7%、37.1% ,其中同时耐 INH(kat G)和 RFP(rpo B)基因突变率为 5 4 .1% ,同时耐三种药的基因突变率为 6 5 .6 %。结论 :PCR- DS法对耐两种或两种以上药物的结核检出率较高 ,与传统药敏试验互相弥补 ,对临床用药有指导意义。     

Comparison of gyrA gene mutations between laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis strains and clinical isolates

To understand the relationship between mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene and drug resistance to ofloxacin, 85 laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis mutant strains and 110 M. tuberculosis clinical isolates, screened by denaturing high-performance liquid chromatography to contain mutations, were analysed for their mutation patterns by sequencing as well as their ofloxacin minimal inhibitory concentrations (MICs). All mutations detected occurred at the codons Ala74, Ala90, Ser91 and Asp94 in all strains. One of the five different forms of missense mutation in Asp94 occurred in 60% of the laboratory-selected strains and 78% of the clinical isolates. However, 53 clinical isolates (48%) and only 2 laboratory-selected strains (2.4%) harboured double point mutations. The mutation Ala74Ser occurred only in the clinical isolates and only in combination with the Asp94Gly mutation. The ofloxacin MIC for the clinical isolates ranged from 0.5microg/mL to 20microg/mL, whilst the MICs for the laboratory-selected strains were > or =10microg/mL. The differences in gyrA gene mutation patterns and MICs between the laboratory-selected resistant strains and clinically isolated resistant strains identified here might help to understand the mechanisms involved in fluoroquinolone resistance.