快速检测结核分枝杆菌异烟肼和利福平耐药相关基因mPCR-SSCP建立及应用

目的 建立快速检测结核分枝杆菌异烟肼(INH)和利福平(RFP)耐药相关基因katG、inhA和rpoB突变的多重聚合酶链反应-单链构象多态性(multi PCR-single strand conformational polymorphism analysis,mPCR-SSCP)方法.方法 药敏试验检测134株结核分枝杆菌临床菌株对INH和RFP的耐药性.设计结核分枝杆菌INH和RFP耐药相关katG、inhA和rpoB基因PCR引物,建立mPCR-SSCP技术检测上述菌株katG、inhA和rpoB基因的突变,同时采用PCR直接测序技术(PCR-DS)检测上述基因片段突变情况,并对上述3种方法检测结果进行分析和比较.结果 134株临床菌株均含有katG、inhA和rpoB基因,其中42株(31.3％)对INH耐药、45株(33.6％)对RFP耐药.mPCR-SSCP和PCR-DS检测结果显示,92株INH敏感菌株katG和inhA基因均未发生突变,检测特异性均为100％;89株RFP敏感菌株中rpoB基因分别有2株和1株检测出突变,检测特异性分别为97.8％和98.9％;42株INH耐药菌株中分别有33株和36株katG和/或inhA基因突变,检测灵敏度分别为78.6％和85.7％;45株RFP耐药菌株中rpoB基因分别有41株和43株发生突变,检测灵敏度分别为91.1％和95.6％.结论 本研究建立的mPCR-SSCP能快速、简便、特异,并有一定的敏感性检测结核分枝杆菌异烟肼和利福平耐药相关基因katG、inhA和rpoB突变,具有临床应用前景.     

结核分枝杆菌耐利福平rpoB基因突变的序列分析

目的探讨结核分枝杆菌耐药基因rpoB突变与利福平耐药性的关系。方法采集81例临床标本,分离结核分枝杆菌菌株,PCR扩增rpoB基因及测序,并与利福平药物敏感试验结果比较。结果27例耐利福平菌株中,20株发生rpoB基因突变(74.1%),突变位点包括531和526在内的7个位点10种突变类型,并发现新的突变位点和突变形式;54株敏感株中,1株发生突变(1.9%)。结论利福平耐药rpoB基因突变有一定区域性,为发展快速的基因诊断技术检测耐多药结核病奠定了基础。     

显色法芯片检测结核分枝杆菌利福平和异烟肼耐药基因

目的建立显色法芯片检测结核分枝杆菌耐药基因的方法。方法设计4对地高辛标记引物,扩增结核分枝杆菌rpoB、katG、inhA和ahpC 4个基因部分片段,根据结核分枝杆菌利福平(RFP)和异烟肼(INH)4条耐药相关基因上8个位点的25种单核苷酸多态性设计探针制作芯片,扩增产物与芯片杂交,显色法判断结果;用该法检测46株结核分枝杆菌临床分离株。结果扩增产物琼脂糖电泳可见4条大小分别为165、181、245、315 bp DNA条带;结核分枝杆菌菌液浓度为1．6×103／ml时,该法仍可检测到各位点野生及突变信号;19种非结核分枝杆菌标准株和9种非分枝杆菌标准株扩增产物无DNA条带,与芯片杂交亦无信号,H37Rv结核分枝杆菌标准株芯片检测各位点均为野生型;5株结核分枝杆菌临床分离株重复检测5次,结果完全一致;6份PCR产物的测序结果与芯片检测结果完全一致;46株结核分枝杆菌临床分离株中,RFP耐药株28株,芯片检出突变株24株,突变率为85．7％,RFP敏感株18株,芯片检出突变株2株。INH耐药株31株,芯片检出突变株20株,突变率为64．5％,INH敏感株15株,芯片检出突变株4株。结论显色法芯片检测结核分枝杆菌耐药基因具有较高的敏感性和特异性,无需特殊仪器设备,有一定的推广应用价值。     

基因型分析法快速检测耐药结核分枝杆菌

目的 探讨基因型分析法检测结核分枝杆菌异烟肼和利福平耐药性的价值.方法 采用多重PCR和线性探针反向膜杂交法来检测异烟肼耐药基因katG S315T和inhA C-15T突变以及利福平耐药基因rpoB D516V,H526Y,H526D,S531L突变来判断78株结核分枝杆菌临床分离株的耐药性,并与金标准L-J固体培养基药敏法以及BACTEC960液体培养药敏法进行对比分析.结果 基因型分析法在6h内可以完成;结核分枝杆菌常规药敏检测需要3个月.与后者相比,基因型分析法检测异烟肼耐药的敏感性和特异性分别为89％ （16/18）和99％ （77/78）;检测利福平耐药的敏感性和特异性均为100％（13/13,78/78）.结论 基因型分析法检测结核菌耐药性快速准确,对耐药结核病的早期诊断和治疗很有帮助,有望在临床实验室广泛开展.     

等位基因特异多重PCR快速检测结核分枝杆菌异烟肼耐药性研究

了解北京地区异烟肼（INI-I）耐药结核分枝杆菌（MTB）的分子特点,评价等位基因特异多重PCR在INH耐药性快速检测中的应用价值。选取102株北京地区MTB临床分离株,首先分别采用PCR—RFLP和反向线性杂交技术（RLB）检测耐药相关基因katG和inhA突变,然后运用等位基因特异多重PCR方法同时检测katG和inhA基因突变,并与PCR—RFLP和RLB分别检测的结果进行比较。采用等位基因特异多重PCR检测发现,INH耐药株中60．3％（35／58）存在katG315突变,13．8％（8／58）发生了inhA突变,两者同时突变率为6．9％（4／58）,INH敏感株中没有发现katG315突变,而inhA突变率为18．2％（8／44）。上述检测结果与PCR-RFLP和RLB分别检测的结果完全一致,且等于两种方法结果之和,提示等位基因特异多重PCR可完全取代PCR—RFLP和RLB。北京地区INH耐药菌株以katG315 AGC→ACC突变为主;联合检测katG315和inhA基因可提高INH耐药株的检出率。等位基因特异多重PCR操作简便,结果易于判断,可作为临床MTB菌株中INH耐药性快速检测的辅助手段。     

M/XDR-TB的快速分子检测和耐药特征分析

目的 使用分子线性探针杂交技术结合仪器法液体快速培养分析耐多药( multidrug resistant,MDR)及广泛耐药(extensively drug resistant,XDR)结核分枝杆菌(Mycobacterium tuberculosis,MTB)的耐药基因和表型特征.方法 运用GenoType MTBDR试剂盒检测M/XDR-TB菌株中各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一线及二线抗结核药物的敏感性.结果 (1)94株MDR-TB经MGIT960检测,乙胺丁醇(ethambutol,EMB)、阿米卡星(amikacin,AMK)、氧氟沙星(ofloxacin,OFX)及莫西沙星(moxifloxacin,MFX)的耐药率分别为36.2%、17.0%、54.3%和55.3%.XDR-TB检出率为13.8%.(2)以MGIT960药敏结果作为参考标准,94株MDR-TB中,GenoType MTBDRplus检测MTB对异烟肼(isoniazid,INH)、利福平(rifampin,RFP)耐药的符合率分别为86.2%和95.7%;GenoType MTBDRsl检测MTB对EMB、AMK、OFX及MFX耐药的敏感性分别为47.1%、81.3%、94.1%、94.2%;特异性分别为75.0%、98.7%、90.7%、92.9%.(3)rpoB基因突变中以S531L最多;INH耐药主要由katG基因发生突变导致,以S315T1类型居多;gyrA突变位点主要集中在第94位密码子.所测菌株中有23例为复合耐药,7例为未知突变.结论 M/XDR-TB中,INH、RFP、AMK、OFX及MFX耐药株大多分别是由katG、rpoB、rrs及gyrA突变导致,乙胺丁醇与embB之间的耐药关联性相对较低.GenoType MTBDR试剂盒检测M/XDR-TB敏感性和特异性较好,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.     

HRCA技术联合DNA芯片检测结核分枝杆菌利福平耐药基因突变的初步分析

目的 对超分支滚环扩增技术(hyperbranched rolling cycle amplification, HRCA)技术联合DNA芯片检测结核分枝杆菌利福平耐药基因突变进行初步分析.方法 采用直接涂片检测在2013年至2015年期间搜集的898份痰液样本中结核分枝杆菌情况,同时采用改良罗氏培养法与HRCA技术联合DNA芯片法对阳性样本进行鉴定,并对利福平耐药基因进行初步分析.结果 989份检测的痰液样本中361份为阳性,涂阳率为40.2%.HRCA技术联合DNA芯片法阳性率为98.0%,显著高于改良罗氏培养法(35.2%),且差异具有统计学意义(P<0.05).通过HRCA技术联合DNA芯片法发现85株结核分枝杆菌对利福平耐药,其中单纯rpoB 基因突变比例为36.5%(31/344);katG基因与rpoB基因均突变比例为60.0%(51/344);inhA基因与rpoB基因突变比例均为3.5%(3/344);且二位点联合突变及单位点突变率分别为89.4%(76/85)与10.6%(9/85).结论 HRCA技术联合DNA芯片法能够有效快速检出结核分枝杆菌突变基因,rpoB基因突变为主要结核分枝杆菌利福平耐药的基础,且突变呈现多样性.     

结核分枝杆菌临床分离株异烟肼耐药相关基因突变的分子特征

目的 阐明结核分枝杆菌异烟肼（INH）耐药相关基因突变特征.方法 对137株结核分枝杆菌临床分离株（耐异烟肼菌株87株,异烟肼敏感菌株90株）的9个结构基因furA、katG、inhA、kasA、Rv0340、iniB、iniA、iniC和efpA以及两个调控区oxyR-ahpC基因间隔区和mabA-inhA启动子进行DNA片段扩增及序列分析.结果 82株（94.3%）INH耐药分离株的katG基因存在突变,其中katGSer315Thr突变占优势（55.2%）.50株INH敏感的分离菌katG的463密码子没有突变.35株（40.2%）INH耐药的分离株katG的463有突变.87株INH耐药株中,20株（23.0%）的katG基因存在两重突变.13株（14.9%）分离菌inhA基因的启动子区存在突变,4.6%的分离菌有inhA结构基因突变,11.5%oxyR-ahpC基因间区存在突变.iniBAC区域和efpA中发现耐药性关联突变.结论 研究证实多个基因突变与异烟肼耐药之间的关系,并且为阐明结核分枝杆菌耐药机制提供线索.     

噬菌体生物扩增法测定痰标本结核分枝杆菌利福平耐药性

目的建立快速测定痰标本中结核分枝杆菌利福平（RFP）耐药性的噬菌体生物扩增法，探讨其在临床应用的可行性。方法噬菌体生物扩增法测定362份涂阳痰标本结核分枝杆菌对RFP的耐药性，并与罗氏绝对浓度法结果进行比较，对不符合的样本，用基因芯片的方法分析rpoB基因的突变情况。结果362份涂阳痰标本，培养阳性360份，菌种初步鉴定有17份样本为非结核分枝杆菌，噬菌体生物扩增法RFP耐药为123份，敏感的196份，两法结果一致的为88．6％，如以绝对浓度法药敏结果为标准，则噬菌体生物扩增法测定利福平耐药的敏感性为93-3％，特异性为87．9％，阴性预测值为96．4％，阳性预测值为78．9％，准确性为89．7％，涂阳等级（1＋～3＋）与实验的有效性相关。结论噬菌体生物扩增法测定痰标本利福平的耐药性只需2d，可作为快速筛诜方法府用千临床．     

刃天青法检测青蒿琥酯对耐药结核分枝杆菌药物敏感性初步研究

目的 了解青蒿琥酯(artesunate,ASN)单独或联合利福平(rifampicin,RFP)、异烟胼(isoniazid,INH)、链霉素(streptomycin,SM)、氧氟沙星(ofloxacin,OFLX)等抗结核药物对耐药结核分枝杆菌的抑制作用,为耐药结核的治疗寻找新的有效药物提供依据.方法 采用刃天青法检测ASN对耐药结核分枝杆菌的抑制作用,并采用ASN联合RFP、INH、OFLX、SM对耐药结核分枝杆菌抗结核药物MIC研究.结果 本研究测试的14株耐药菌株中,7株MIC值为256μg/mL,6株MIC值为128μg/mL,1株MIC为512μg/mL,MIC50为256μg/mL,MIC90为256μg/mL.16株敏感株中,MIC50为128μg/mL,MIC90为256μg/mL.两者差异无统计学意义(P＞0.05).此外,当ASN终浓度为32 ～ 128μg/mL时,能明显改善耐药MTB对RFP、INH、OFLX、SM等抗结核药物的敏感性.结论 ASN对抗耐药结核分枝杆菌作用可能甚微,但其能协同或逆转抗结核药物改善耐药MTB的药物敏感性,可能成为耐多药结核治疗备选新药.     

Sequence analysis of rpoB mutations in rifampin-resistant clinical Mycobacterium tuberculosis isolates from Turkey

Drug-resistant tuberculosis is a serious problem throughout the world. Resistance to Rifampicin (RIF) is mainly caused by the mutations in the rpoB gene coding the beta-subunit of RNA polymerase. In this study, we aimed to detect the distribution of rpoB gene mutations in 80 RIF-resistant clinical Mycobacterium tuberculosis (MTB) isolates from Turkey. The rpoB gene was amplified by PCR and mutations leading to RIF resistance were determined by automated sequence analysis. A total of 72 of the 80 isolates (90%) were found to carry mutations in the amplified region, whereas eight isolates (10%) carried no mutations. Overall, 24 different missense mutations affecting 14 codons, and two deletion mutants were identified. Nine new mutations, six in the hot-spot region and three outside this region, were found. The codon numbers of the most frequently encountered mutations were 531 (51.4%), 526 (18.1%), 516 (13.9%), and 513 (12.5%). As a result, 90% of the RIF-resistant MTB isolates from the Turkish patients were found to carry a mutation in the rpoB gene, Ser531Leu being the most frequent one. Although molecular methods identify mutations leading to RIF resistance very quickly, results of the antimycobacterial susceptibility tests must be taken into consideration for the patients carrying no mutations in this region.     

Molecular characteristics of rifampin- and isoniazid-resistant mycobacterium tuberculosis strains isolated in Vietnam

Molecular characterization of the drug resistance of Mycobacterium tuberculosis strains with different origins can generate information that is useful for developing molecular methods. These methods are widely applicable for rapid detection of drug resistance. A total of 166 rifampin (RIF)- and/or isoniazid (INH)-resistant strains of M. tuberculosis have been isolated from different parts of Vietnam; they were screened for mutations associated with resistance to these drugs by sequence analysis investigating genetic mutations associated with RIF and INH resistance. Seventeen different mutations were identified in 74 RIF-resistant strains, 56 of which (approximately 76%) had mutations in the so-called 81-bp "hot-spot" region of the rpoB gene. The most common point mutations were in codons 531 (37.8%), 526 (23%), and 516 (9.46%) of the rpoB gene. Mutations were not found in three strains (4.05%). In the case of INH resistance, five different mutations in the katG genes of 82 resistant strains were detected, among which the nucleotide substitution at codon 315 (76.83%) is the most common mutation. This study provided the first molecular characterization of INH and RIF resistance of M. tuberculosis strains from Vietnam, and detection of the katG and rpoB mutations of the INH and RIF-resistant strains should be useful for rapid detection of the INH- and RIF-resistant strains by molecular tests.     

Use of the genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis complex isolates

A commercially available DNA strip assay (Genotype MTBDR; Hain Lifescience, Nehren, Germany) was evaluated for its ability to detect mutations conferring resistance to rifampin (RMP) and isoniazid (INH) in clinical Mycobacterium tuberculosis complex isolates. A total of 103 multidrug-resistant (MDR; i.e., at least resistant to RMP and INH) and 40 fully susceptible strains isolated in Germany in 2001 in which resistance mutations have been previously defined by DNA sequencing and real-time PCR analysis were investigated. The Genotype MTBDR assay identified 102 of the 103 MDR strains with mutations in the rpoB gene (99%) and 91 strains (88.4%) with mutations in codon 315 of katG. All 40 susceptible strains showed a wild-type MTBDR hybridization pattern. The concordance between the MTBDR assay and the DNA sequencing results was 100%. Compared to conventional drug susceptibility testing, the sensitivity and specificity were 99 and 100% for RMP resistance and 88.4 and 100% for INH resistance, respectively. In conclusion, the MTBDR assay is a rapid and easy-to-perform test for the detection of the most common mutations found in MDR M. tuberculosis strains that can readily be included in a routine laboratory work flow.     

深圳地区结核分枝杆菌耐药分离株分子特征与表型特征相关性研究

目的 研究深圳地区2007-2008年分离的结核分枝杆菌耐药株分子特征与表型特征的相关性.方法 参照WHO/IUATLD标准,使用L-J药敏培养基,1%比例法药敏试验筛选针对异烟肼、利福平、链霉素、氧氟沙星、卡那霉素5种药物耐药或敏感的临床分离株,通过PCR扩增经筛选菌株的rpoB、katG、rpsL、rrs1、gyrAB、rrs2基因的相关序列,运用DNAStar和BLASTN进行序列分析,应用二倍稀释法测定其表型最低抑菌浓度(MIC)值.结果 筛得实验菌株123株,其中耐药株73株,全敏感株50株.异烟肼耐药株katG基因突变率为84.6%,突变位点全部为S315T或S315N.利福平耐药株rpoB基因突变率为93.6%,突变位点主要集中在S531L(30/44,68.2%)和H526D(9/44,20.5%)或H526R(1/44,2.3%).链霉素耐药株以rpsl基因突变为主,突变位点为K43R(19/27,70.4%)和K88Q(6/27,22.2%);rrs1基因突变较少见,仅491C→T(2/27,7.4%)及513A→C(1/27,3.7%);2个基因突变率合计为65.9%.氧氟沙星耐药株突变率为100%,以gyrA基因突变为主,突变位点包括D94A(2/11,18.2%)、S91P(4/11,36.4%)和A90V(3/11,27.3%),3个突变位点总突变率为81.8%(9/11);S95T存在于所有氧氟沙星耐药株及部分全敏感株gyrA基因中;gyrB未发现突变.卡那霉索耐药株rrs2基因突变率为61.1%;突变位点为1400 A→C(9/11,81.8%)和1483 G→T(2/11,18.2%).其他突变位点在全敏感株中未发现.临床耐药株同一耐药组别所包含耐药类型不同,相关耐药基因的突变位点相同,其MIC值基本一致;相关耐药基因突变位点不同,其MIC值存在明显差异.结论 结核分枝杆菌耐药基因突变存在一定的地域特性,表型特征因耐药基因突变位点不同存在耐药程度差异.     

Results of the use of biochips to determine the drug resistance of Mycobacterium tuberculosis in the Novosibirsk and Tomsk regions

To evaluate the possibility of determining rifampicin resistance using a biochip developed at the Institute of Molecular Biology of the Russian Academy of Sciences (Moscow), 272 rifampicin-resistant isolates of M. tuberculosis, isolated from tuberculosis patients in the Novosibirsk and Tomsk regions in 2000–2005, were investigated. Rifampicin resistance was determined by the method of absolute concentrations on Lowenstein-Jensen medium; mutations in the rpoB gene were determined with the biochip and simultaneously by the SSCP method. Moreover, the variable region of the rpoB gene was sequenced in 60 strains of a randomized sample. In 86% of the cases, the results of the determination of rifampicin resistance by the method of absolute concentrations and with the biochip were in full agreement. The results differed in cases when cultures isolated from a patient contained a mixture of sensitive and resistant strains. Four main codons in which mutations can cause resistance to rifampicin were detected: S531 (76.2%), H526 (7%), D516 (5.6%), and L511 (5.6%). Resistance to isoniazid was also established in the region among 94% of the rifampicin-resistant strains, i.e., they were resistant to a number of drugs. Consequently, the biochip for the determination of rifampicin resistance can also be used to determine multiple-drug-resistant (MDR) strains. The use of the biochip to diagnose rifampicin resistance is significantly simplified if the two steps of the polymerase chain reaction (PCR) are replaced by one-step variant, in which a single-stranded labeled PCR fragment is synthesized according to a “nested PCR” scheme in one test tube.     

Simultaneous identification and typing of multi-drug-resistant Mycobacterium tuberculosis isolates by analysis of pncA and rpoB

In Mycobacterium tuberculosis there is a strong correlation between in-vitro resistance to rifampicin (RIF) and pyrazinamide (PZA) and mutations in rpoB and pncA, respectively. Approximately 50 mutations associated with resistance have been reported for rpoB and 70 for pncA, and, theoretically, many more are possible. Therefore, the identification of rpoB and pncA mutations in M. tuberculosis might be used for the simultaneous determination of resistance and for typing multi-drug-resistant (MDR) strains during possible outbreaks. The present study examined four sensitive and six MDR isolates of M. tuberculosis from Turkey and eight isolates from a nosocomial MDR tuberculosis (TB) outbreak in the UK. Gene mutations were identified by the Innogenetics LiPA rpoB assay or automated sequencing, or both. All the sensitive isolates had rpoB and pncA wild-type genotypes, whereas all the RIF- and PZA-resistant isolates had rpoB and pncA mutations. All four mutations seen in rpoB, but none of the six in pncA, had been reported previously. The rpoB and pncA mutations seen in the Turkish isolates defined six distinct genotypes amongst the six MDR isolates, while standard IS6110 typing discriminated only four. All isolates from the single strain MDR-TB outbreak had identical genotypes. Rapid genotyping was performed on the sputum from a patient who presented 2 years after the initial MDR-TB outbreak and this showed rpoB and pncA genotypes identical to the other outbreak isolates. This result was available within 36 h. The analysis of rpoB and pncA is a rapid and practical means of simultaneously identifying and typing MDR isolates of M. tuberculosis.     

Association of specific mutations in katG, rpoB, rpsL and rrs genes with spoligotypes of multidrug-resistant Mycobacterium tuberculosis isolates in Russia

Most multidrug-resistant (MDR) Mycobacterium tuberculosis isolates in Russia belong to the Beijing or Latino-American and Mediterranean (LAM) spoligotype families. The objective of this study was to investigate possible associations between genotype and the frequencies of mutations that confer drug resistance in a population that has two large families of circulating strains. Spoligotyping, IS6110 restriction fragment length polymorphism typing, and sequencing of the katG and rpoB genes, were performed for 217 consecutive MDR M. tuberculosis isolates from patients. The rpsL and rrs genes were also sequenced for selected streptomycin-resistant isolates. Of the 217 MDR isolates, 99 (46%) belonged to the LAM family, 92 (42%) to the Beijing family, 21 (10%) to the Haarlem family and four (2%) to the T family. There was one unique spoligotype. Mutations in the katG gene were identified in 207 (95%) isolates, all of which had mutations in codon 315. Mutations in the rpoB gene were identified in 200 (92%) isolates; 75% of LAM isolates carried a mutation in codon 516, whereas 71% of Beijing isolates carried a mutation in codon 531. In the 33 isolates resistant to streptomycin 50 mg/L, the 43AGG rpsL mutation was found in 27% of Haarlem, 75% of Beijing and 0% of LAM isolates, and rrs mutations were found in 17% (516C-->T) of Beijing and 100% (513A-->C) of LAM isolates. Overall, there appeared to be a correlation between the genotype and specific mutations conferring resistance to rifampicin or streptomycin in the Beijing and LAM families. The biological implications of this correlation remain to be explored.