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.     

Application of PCR-SSCP in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis

Objective: To study the relationship between the polymorphism of drug resistant gene rpoB and drug resistance against rifampicin(RFP) of M. tuberculosis L-forms, and to evaluate its clinical application. Methods: A total of 52 clinical isolated strains of M. tuberculosis L-forms were collected. rpoB gene polymorphism was analyzed by polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) and conventional antimicrobial susceptibility test (AST). Their results were compared. Results: AST results showed that 38 of 52 clinical isolated strains were drug resistance (73.08％),while PCR-SSCP indicated 65.38％ (32/52) rpoB gene polymorphism. There was no statistic significance(χ2= 2.4914) between the 2 methods. Conclusion:Combined the application of PCR-SSCP with AST in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis may have advantages at earlier diagnosis and guidance of clinical medications.     

PCR-SSCP法检测结核分枝杆菌耐药性

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以及聚合酶链反应-单链构象多态性分析(poly merase chain reaction-single strand cinfomlation polymorphism，PCR-SSCP)方法的临床应用价值。方法：用PCR-SSCP方法检测58株结核分枝杆菌临床分离株katG，rpoB，rpsL基因突变并与常规药敏试验检测结果进行对比。结果：经常规药敏试验检测，58株结核分枝杆菌临床分离株中共有41株耐药，其中，耐异烟肼(INH)为35株，高耐药株27株；耐利福平(RFP)为31株，高耐药株24株；耐链霉素(SM)有31株，高耐药株26株。同时耐3种药物的有21株(51．2％)，耐2种药物的14株(34．1％)，单耐药株6株(14．6％)．PCR-SSCP方法对58株临床分离株katG，rpoB，rpsL基因突变的检测率为40％(23／58)，45％(26／58)，38％(22／58)，其中检出3个基因同时突变的有13株(32％)，2种基因突变的12株(29％)，1种基因突变的有10株(23．8％)．常规药敏试验与PCR-SSCP法检出结核分枝杆菌同时耐3种药物的符合率为61．9％(13／21)，检出耐2种药物的符合率为85．70k，(12／14)，检出耐1种药物的符合率为50％(3／6)．高耐药株中突变率为80．5％(62／77)，低耐药株中突变率为60％(12／20)．结论：PCR-SS-CP方法对耐2种以上药物的结核杆菌检出率较高，且耐药基因突变率随着耐药浓度增高而增高。将PCR-SSCP法与药敏试验联合应用可互相弥补，已成为临床指导用药的好方法。     

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。     

耐异烟肼结核分枝杆菌及其katG、inhA基因的突变

目的 探讨耐异烟肼结核分枝杆菌及其katG与inhA基因突变的特征,为临床选择抗结核药物治疗提供实验室参考。方法 回顾性分析2015年1月1日-2017年1月1日在上海交通大学附属同仁医院海南分院的260例患者标本,对213例进行结核分枝杆菌分离培养,对47例进行博奥芯片法检测,再对213例培养所得菌株进行博奥芯片检测,对博奥芯片检测的47例痰标本进行结核分枝杆菌分离培养;对101株结核分杆菌进行katG与inhA基因检测。结果 培养法和博奥芯片结核分枝杆菌检出率分别为36.6%（78/213）和48.9%（23/47）;培养法和博奥芯片耐多药结核分枝杆菌检出率分别为41.0%（32/78）和47.8%（11/23）;博奥芯片法和比例法耐异烟肼符合率98.7%(77/78);男性结核分枝杆菌阳性率45.9%高于女性阳性率29.2%;检测katG基因和inhA基因,3个基因区域都发生突变,突变发生率大小依次为katG315（AGC→ACC）密码子(32株,54.2%)、katG315（AGC→AAC）密码子(16株,27.1%)、inhA-15（C→T）(10株,16.9%)。24株(23.8%,24/101)对异烟肼无突变但对利福平发生突变;43株也同时耐利福平(42.6%,43/101)。突变频率较高的突变位点是315,突变频率为81.4%(48/59),1株(1.7%,1/59)为双位点联合突变且突变频率最低。结论 结核分枝杆菌katG和inhA基因突变与耐INH相关,这为临床及时准确诊断、及早使用抗结核药物联合治疗提供了帮助。     

结核分支杆菌耐利福平耐药基因检测研究

目的评价应用DNA序列分析方法和聚合酶链反应-单链构象多态性(PCR-SSCP)技术检测rPOB基因突变在结核分支杆菌耐利福平(RFP)耐药性测定中的应用价值.方法采用DNA序列分析法和PCR-SSCP法对80株结核分支杆菌临床分离株(其中药物敏感株32株,耐RFP或含耐RFP耐多药株48株)rpoB基因核心区域的突变情况进行检测.结果以结核分支杆菌H37RV为对照,所有32株药物敏感株的rPOB基因均无突变.用DNA序列分析方法检测48株耐药株中44株发生突变,敏感性为91.7%(44/48),其中最常见的突变位点是531位丝氨酸和526位组氨酸.PCR-SSCP检测48株耐RFP分离株中,45株rPOB基因SSCP图谱异常,其敏感性为93.1%(45/48).结论DNA序列分析可指导临床用药,PCR-SSCP可快速检测结核分支杆菌RFP耐药性.     

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.     

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。     

PCR—SSCP快速检测脊柱结核耐药基因

目的 了解临床脊柱结核耐药情况 ,探讨耐药基因PCR-SSCP对临床的应用价值.方法 43例脊柱结核患者结核肉芽组织应用BacT/ ALERT 3D结核分支杆菌快速培养系统培养,所得临床分离株行药敏试验,对耐药基因katG、r p s L行PCR-SSCP.结果 43例结核肉芽组织标本培养后19例阳性,其中6株存在不同程度耐药 ,总耐药率为31%.耐异烟肼株 katG突变率为 10% ,耐链霉素株 rpsL突变率为21% , 耐药基因检测平均用时为14h.结论 PCR-SSCP可快速检测脊柱结核耐药基因,与常规药敏试验结合可更能准确地反应脊柱结核患者的耐药情况.     

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。     

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。     

痰液分离结核分枝杆菌202株耐药性分析

目的:了解结核分枝杆菌对几种常用抗结核药的耐药性.方法:采用绝对浓度法,将每种药品配制高低2种浓度,加至改良罗氏培养基中,再接种临床分离的结核杆菌菌株,观察细菌的生长情况.结果:202株临床分离结核分枝杆菌对异烟肼、链霉素硫酸盐、乙胺丁醇、利福平、对氨基水杨酸的耐药率分别为25.7%、22.3%、5.9%、32.2%、11.4%;全部耐药菌株约占3.5%.结论:乙胺丁醇可作为结核病治疗的有效药物之一,结核分枝杆菌对几种抗结核药的耐药率有不同程度的提高,临床工作中,应增加药敏试验药物品种.     

Sequence analysis on drug-resistant rpoB gene of Mycobacterium tuberculosis L-form of isolated from pneumoconlosls workers

To study the characteristics of drug-resistant genetic mutation of rpoB on coal workers＇ pneumoconiosis complicated with L-form of Mycobacterium tuberculosis. Methods： A total of 42 clinical isolated strains of Mycobacterium tuberculosis L-forms were collected, including 31 drug-resistant strains. Their genomes DNA were extracted, the target genes were amplified by PCR, and the hot regions in the rpoB gene were analyzed by automated DNA sequenator. Results： No mutation of rpoB gene was identified in 11 rifampicin-sensitive strains while conformation changes were found in 31 rifampicin-resistant strains. The mutation rate was 93.55% （29/31） in resistant strains, mainly concentrated in codon 531 （51.6%, 16/31） and 526 （32.26%, 10/31）. Base substitutions happened, including 27 unit point mutation and 2 two point mutation. The mutation of codon 516 that new found wasn＇t reported by internal and overseas scholars. Conclusion： The substitution of highly conserved amino acids encoded by rpoB gene results in the molecular mechanism responsible for rifampicin resistance in Mycobacterium tuberculosis L-forms. It also proves that rpoB gene is diversiform.     

结核分枝杆菌异烟肝耐药基因突变的研究

目的：了解我国结核分枝杆菌耐异烟肼（INH）分离株katG基因突变情况,探讨快速检测结核分枝杆菌耐药基因型的分子药敏检测方法。方法：用PCR单链构象多态性（PCR－SSCP）和PCR直接测序法（PCR－DS）检测30株结核分枝杆菌临床分离株的katG基因。结果：以结核分枝杆菌H37Rv标准株对照观察所有INH敏感株的PCR－SSCP和PCR－DS图谱,均未发现异常;而在12株INH耐药株中,有4株无PCR－SSCP和PCR－DS图谱异常,占INH耐药株的33．4％;有5株在94位发生核苷酸错义突变,占INH耐药株41．7％,有3株在96位发生核苷酸同义突变,占INH耐药株的25％。结论：多数结核分枝杆菌对INH是由于其katC基因突变所致,可先用PCR－SSCP筛选突变株,再用PCR－DS方法测定其突变位点,达到快速检测结核分枝杆菌INH耐药基因型的目的。     

结核杆菌利福平耐药与rpoB基因突变的相关性研究

目的：分析西安地区耐利福平（RIF）结核杆菌临床分离株rpoB基因突变的特点,探讨快速检测结核杆菌耐RIF基因型的方法．方法：对120株西安地区收集的结核杆菌临床分离株（药敏结果为耐RIF81株,对RIF敏感39株）,分别采用聚合酶链反应一单链构象多态性（PCR-ssce）和等位基因特异性多重聚合酶链反应（MAS—PCR）检测rpoB基因突变情况．结果：PCR—SSCP检测,耐RIF菌株中rpoB基因突变发生率为85％（69／81）,39株敏感株中仅有1株发生突变,突变率为2％（1／39）．MAS—PCR检测,耐RIF菌株中rpoB基因突变发生率为93％（75／81）,39株敏感株中未发现rpoB基因突变;531,516和526位点突变的比例分别为45％（34／75）,24％（18／75）和20％（15／75）．结论：西安地区结核杆菌临床分离株耐RIF与rpoB基因突变相关,MAS-PCR检测rpoB基因可作为临床耐RIF结核杆菌的简单快速准确的早期诊断方法．     

结核分支杆菌异烟肼耐药的分子机制及其快速鉴定

目的：探讨快速检测结核分支杆菌异烟肼耐药的分子药敏方法。方法：用聚合酶链反应-单链构象多态性(PCR-SSCP)检测了20株异烟肼(INH)敏感的结核杆菌临床分离株，20株INH耐药株的katG基因，随后用SSCP方法鉴定扩增产物有无突变，以结核分支杆菌H37RV作对照。结果：所有INH敏感株均观察到katG基因扩增产物，20株INH耐药株中19株观察到katG基因扩增产物。以H37RV标准株为对照，20株敏感株SSCP带谱与对照相同；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耐药katG基因突变或缺失的阳性率为60％。结论：多数结核分支杆菌耐INH是由于其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支杆菌INH耐药的目的。     

DNA微阵列芯片法检测结核分枝杆菌耐异烟肼基因的研究

目的采用DNA微阵列芯片法检测结核分枝杆菌INH耐药基因,为临床诊治提供实验依据。方法应用DNA微阵列芯片法和改良罗氏培养加比例法药敏试验对疑似结核病患者的150份痰液标本进行检测,并对检测的结果进行比较分析。结果 150份痰液标本的涂阳率为60.0%(90/150),DNA微阵列芯片法检测到结核分枝杆菌阳性率为62.7%(94/150),改良罗氏培养法阳性率为60.0%(90/150),比例法药敏试验异烟肼的耐药率为43.9%(36/82),异烟肼的kat G/inh A基因总突变率为35.1%(33/94);以改良罗氏培养加比例法药敏试验为标准,DNA微阵列芯片法检测结核分枝杆菌阳性、异烟肼耐药性和敏感性的符合率分别为97.7%、86.1%和100.0%。结论 DNA微阵列法可快速准确地检测大部分疑似结核病临床标本的kat G和inh A基因突变,可用于临床耐药性的检测从而指导临床用药,也可用于临床诊断中的推广。     

荧光PCR熔解曲线法对痰样本结核分枝杆菌耐药性检测价值及耐药特征分析

目的：评估荧光PCR熔解曲线法（熔解曲线法）检测肺结核患者涂阳痰样本结核分枝杆菌（MTB）对一线抗结核药物耐药性价值及耐药特征分析。方法：收集涂阳且培养阳性肺结核患者痰样本和相应MTB菌株各142例,采用熔解曲线法检测痰样本MTB对抗结核药物链霉素（SM）、异烟肼（INH）、利福平（RFP）、乙胺丁醇（EMB）耐药情况,同时采用液体药敏法对相应MTB菌株耐药性检测,对两种检测结果行方法学比较。结果：以液体药敏法为标准,熔解曲线法检测痰样本MTB对4种抗结核药物耐药性结果差异无统计学意义（P>0.05）,熔解曲线法检测SM、INH、RFP、EMB耐药性的敏感度分别为：78.57% (11/14),70.00% (14/20),100.00% (12/12),71.43% (5/7);特异度分别为：92.97%(119/128),96.72%(118/122),97.69%(127/130),100%(135/135);两者符合率分别为：91.55%(130/142),92.96%(132/142),97.89%(139/142),98.59%(140/142);Kappa值分别为：0.601,0.696,0.877,0.826。结论：与传统液体药敏检测相比,熔解曲线法检测痰样本MTB对抗结核药物SM、INH、RFP、EMB的耐药性效能一致,特异度和符合率均较高。该法对结核患者涂阳痰样本MTB耐药性检测有较好临床参考价值。     

713株结核分枝杆菌耐药性分析

目的分析2005年以来我中心肺科就诊的肺结核病人结核分枝杆菌耐药趋势,为结核病的防治提供依据。方法应用改良罗氏培养基间接药敏实验绝对浓度法检测结核菌的耐药性情况。结果1754株结核分枝杆菌耐药713株,总耐药率为41．0％;耐多药（MDR）111株,耐多药率为6．3％。耐单药顺位从高到低依次为SM（26．1％）。RFP（25．3％）,INH（14．1％）,EMB（10．3％）。各年龄组耐药及耐多药均为青年组〉中年组〉老年组。结论结核分枝菌抗药性呈增强趋势,应加强监测,指导防控工作和临床合理用药。     

结核分支杆菌rpoB基因突变和耐利福平的相关性研究

研究结核分支杆菌ｒｐｏＢ基因突变及其与利福平（ＲＦＰ）耐药性的关系,建立快速检测耐药基因型的分子药敏试验方法。用ＰＣＲ—ＳＳＣＰ分析结核分支杆菌ｒｐｏＢ基因。结果显示ＰＣＲ扩增ｒｐｏＢ基因为属特异性。８１株结核分支杆菌的ＰＣＲ－ＳＳＣＰ图谱与Ｈ３７Ｒｖ为对照,３５株敏感菌仅有一株位置有区别;４６株耐药菌有４２株有明显区别;检测阳性率为９１．３％;特异性９７．１％。证明ｒｐｏＢ基因突变是结核分支杆菌耐利福平的主要机理,ＰＣＲ—ＳＳＣＰ可简便快速地检出ｒｐｏＢ基因的突变,有助于结核分支杆菌耐药性的快速检测和研究。