应用专一性寡核苷酸微阵列可靠地检测结核分枝杆菌利福平耐药性

DNA微阵列代表聚合酶链反应产物诊断测序的发展方向 .根据结核分枝杆菌rpoB基因利福平抗药性决定区域内点突变及其它重排的特征 .研制一种快速地鉴定结核分枝杆菌利福平耐药菌株的中等密度微阵列方法 .利福平抗药性通过使荧光标记扩增遗传物质与微阵列杂交测定 .检测5 3株利福平耐药结核分枝杆菌和 15株利福平敏感结核分枝杆菌 .微阵列方法的检测结果与药物敏感性试验和DNA测序结果完全一致 .临床标本PCR扩增后仅 1 5h可检出利福平耐药临床分离株 .表明寡核苷酸微阵列是高效的、专一性的方法 ,可作为检测利福平抗药性的快速方法以弥补传统培养方法的不足     

PCR-SSCP检测结核分枝杆菌利福平耐药相关基因rpoB突变

目的 建立聚合酶链反应-单链构象多态性(PCR-SSCP)技术快速检测结核分枝杆菌利福平(RFP)耐药相关基因rpoB突变.方法 设计结核分枝杆菌RFP耐药相关rpoB基因PCR引物,建立PCR-SSCP技术检测临床菌株rpoB基因的突变导致的运动变位,同时采用PCR直接测序(PCR-DS)技术检测rpoB基因突变,并对上述方法检测结果进行分析和比较.结果 84株临床菌株均含有rpoB基因;PCR-SSCP和PCR-DS检测结果显示,56株RFP敏感菌株中rpoB基因分别有3株和2株检测出突变,检测特异性分别为94.6％ (53/56)和96.4％(54/56);28株RFP耐药菌株中rpoB基因分别有27株和28株发生突变,检测灵敏度分别为96.4％和100％.结论 本研究建立的PCR-SSCP技术能快速、简便、特异、敏感地检测结核分枝杆菌利福平耐药基因rpoB突变,具有临床应用前景.     

基于PARMS技术的结核分枝杆菌利福平耐药基因rpoB分子标记的开发

文中旨在建立结核分枝杆菌利福平耐药基因rpoB的荧光分子标记,为分子药物敏感性试验提供简便、可靠的基因分型检测方法。对比分析利福平耐药菌株rpoB基因531、526、516、511、513等氨基酸位点的基因突变与敏感菌株中等位基因的序列差异,结合PARMS技术 (Penta-primer amplification refractory mutation system),建立rpoB基因的荧光分子标记。利用其对104例结核分枝杆菌临床分离株进行检测,经Sanger测序验证,正确率100%。并采用比例法药敏试验对这104份样本进行了利福平耐药性鉴定,与分子标记结果相符率为94.23%。结果表明,分子标记有较强可靠性,能检出表型药敏无法测出的低浓度耐药样本 (511/533单位点突变)。建立的11组荧光分子标记能覆盖92%–96%的利福平耐药菌株rpoB基因突变类型,为快速检测结核分枝杆菌利福平耐药提供新思路。     

淋病流行株多重耐药性与mtrRCDE外排系统的关系

为了探讨mtrRCDE外排系统与淋病流行株多重耐药性的关系 ,应用K B法和琼脂稀释法从湛江地区分离出 6 2株淋球菌多重耐药株。利用煮沸法提取细菌DNA ,PCR扩增mtrR基因 ,并对扩增产物测序 ,比较敏感株与多重耐药株的差异。结果显示 :5株敏感株和 2株耐青霉素淋球菌无mtrR的突变 ,10株多重耐药株均有mtrR基因的突变 ,表现为第 12 4位和第 139位G→A。从而提示 ,mtrRCDE外排系统与淋病流行株的多重耐药性密切相关。     

深圳地区结核分枝杆菌耐利福平株rpoB基因突变分布

目的:调查深圳地区结核分枝杆菌耐利福平(RFP)株rpoB基因突变的分布情况,建立结核分枝杆菌耐药基因快速检测的方法.方法:对55株结核分枝杆菌临床分离株的rpoB基因280个碱基(包括其核心区75个碱基)应用PCR-直接测序法(PCR-DS)测定序列,其中耐利福平株51株,敏感株4株.结果:4株敏感株无突变,92.2%(47/51)耐利福平临床分离株存在rpoB基因突变.基因突变导致531位氨基酸突变率为41.2%(21/51);导致526位氨基酸突变率为29.4%(15/51);导致516位氨基酸突变率为13.7%(7/51).联合突变发生率为2.0%(1/51).未检测到发生缺失或插入碱基突变的菌株.结论:深圳地区结核分枝杆菌耐利福平株发生rpoB基因突变最常见的是531位丝氨酸、526位组氨酸和516位天冬氨酸的基因突变,三者突变率之和为84.3%(43/51).PCR-DS方法可快速测定结核分枝杆菌RFP耐药基因突变.     

耐亚胺培南铜绿假单胞菌的耐药性特征及oprD基因突变分析

为了分析亚胺培南耐药铜绿假单胞菌(IMPRPAE)临床分离株的耐药性及oprD基因变异情况,本研究收集了2014年1月至2017年12月临床标本中分离的35株IMPRPAE,采用VITEK2-compact系统分析IMPRPAE的耐药性;PCR法扩增基因oprD并测序分析其序列突变类型。IMPRPAE共15株,其中1株oprD基因PCR扩增阴性。余14株阳性菌株进行oprD基因测序发现其中1株无突变,11株有框码移位(其中有2株发现携带插入序列ISPpu21, IS1394),2株有终止密码子提前出现。本研究中铜绿假单胞菌oprD基因突变是亚胺培南耐药的主要原因。     

耐药结核分枝杆菌感染动物模型所用菌株的筛选

本研究通过小鼠体内实验检测我国部分地区结核分枝杆菌耐药菌株的毒力,以筛选耐药结核分枝杆菌感染动物模型所用菌株。收集从我国部分省份98例结核病患者痰培养液中分离出的结核分枝杆菌,用比例法药敏试验进行结核分枝杆菌一线和二线药物的药敏试验,筛选出对二线药物敏感而对一线药物利福平或异烟肼耐药或敏感的菌株,然后进行小鼠体内毒力实验,对异烟肼耐药相关基因katG和利福平耐药相关基因rpoB测序并进行基因突变分析。从98株菌中筛选出药物敏感谱清晰的40株,进行小鼠体内毒力实验。结果显示,共35株半数死亡时间≤H37Rv的半数死亡时间,其中18株耐利福平合并耐异烟肼、5株单耐利福平,7株单耐异烟肼、5株对利福平和异烟肼均敏感。通过小鼠毒力研究,分别筛选出基因背景清晰,半数死亡时间≤7d的耐利福平合并耐异烟肼的菌株1株、半数死亡时间≤7d单耐利福平和异烟肼的菌株各1株,作为耐药结核分枝杆菌感染小鼠模型所用菌株及进一步进行豚鼠等其他动物模型感染用候选菌株。     

基于锁核酸及等位位点特异性扩增技术的KRAS基因突变检测荧光定量PCR方法研究

基于等位位点特异性扩增的原理,设计锁核酸修饰KRAS基因突变特异性扩增引物,结合封阻探针技术,建立检测KRAS基因突变的荧光定量PCR方法。结果发现,锁核酸修饰的引物及探针可显著提高等位位点特异性扩增技术用于复杂样本中的微量基因突变检测的敏感度,该技术检测KRAS基因突变的敏感性可达0.01%~0.1%。进一步用建立的荧光定量PCR方法检测52例结直肠癌患者血浆标本,并用DNA测序法作为对照,同时用健康人血浆标本建立阴性检测结果判读标准,以初步评价该方法应用于循环DNA中KRAS基因突变检测的可行性。结果发现结直肠癌患者KRAS基因突变主要是G12C、G12A和G12R,而且q PCR法的阳性检出率为46.15%,高于DNA测序法(13.46%),阴性结果与DNA测序法的符合率为100%。此外,结直肠癌患者外周血KRAS基因的突变检出率与文献报道组织标本中的突变检出率及常见突变类型基本相符。上述结果说明该方法检测循环肿瘤DNA(circulating tumor DNA,ct DNA)具有较高的可靠性,可以用于肿瘤患者循环血液中KRAS基因突变的检测。     

结核分枝杆菌利福平耐药性的研究进展

本旨在阐明结核分枝杆菌耐利福平分离株rpoB基因突变的规律,以及rpoB基因突变与利福平最低抑菌浓度(MIC)的关系。结核分枝杆菌的rpoB基因突变是引起利福平耐药性的主要原因,耐利福平分离株的rpoB基因突变主要集中在507～533位密码子的81bp的区域内,约80％的菌株发生531位或526位密码子突变。不同类型rpoB基因突变的结核分枝杆菌对利福平的耐受性也不同,通常发生531位密码子突变的菌株的MIC≥64μg／ml。     

头孢西丁耐药肺炎克雷伯菌AmpC基因和ESBLs检测及耐药性分析

目的了解深圳地区头孢西丁耐药肺炎克雷伯菌头孢菌素酶(AmpC酶)基因型分布、产超广谱β-内酰胺酶(ESBLs)的情况及其耐药特点。方法收集深圳地区三家大型综合医院临床标本分离对头孢西丁耐药的肺炎克雷伯菌73株。用碱裂解法提取菌株的质粒,采用多重PCR扩增AmpC基因,应用DNA测序确定其基因型。并对所有菌株进行ESBLs表型确证试验;用K-B法对其进行药物敏感试验。结果 48株(65.8%)AmpC基因扩增阳性,经DNA测序显示,其中46株为DHA-1型,1株为CMY-2型,1株同时产DHA-1和CMY-2型;73株肺炎克雷伯菌中49株ESBLs阳性,其中36株AmpC基因和ESBLs均为阳性。AmpC和(或)ESBLs阳性菌株对多数药物的耐药率高于AmpC和ESBLs均阴性者。结论本地区头孢西丁耐药肺炎克雷伯菌质粒AmpC酶检出率高,基因型主要为DHA-1,同时产AmpC酶和ESBLs菌株较常见。     

结核分枝杆菌rpoB基因突变的检测(简报)

结核病主要是由结核分枝杆菌(Mycobacterium tuberculosis)引起的一种慢性传染性疾病。利福平是结核病化疗方案中一个关键性的药物,它在结核病的短程化疗中起着重要的作用。但是,在我国结核菌对利福平的耐药发生率呈上升局势,而通过传统的依赖生物生长的药敏试验方法进行结核菌对利福平耐药性检测所需时间较长(4-8周),不能满足临床早期开展有效化疗的需要,所以迫切需要建     

Evaluation of biochips for the rifampin resistance detection of M. tuberculosis in strains isolated at the Novosibirsk and Tomsk regions

Recently in Russia biochips for rifampin resistance detection of M. tuberculosis were developed. To investigate the conformity between rifampin resistance results determined both by the routinely used absolute concentration method and USING the biochips, 272 DNA samples of M. tuberculosis isolated from TB patients at Novosibirsk and Tomsk regions in 2000-2005 were analyzed. The biochip can detect 30 mutations in rpoB gene. The mutations were also tested using the single stranded conformational polymorphism method (SSCP). In addition, 60 DNAs were randomly sampled and sequenced. The results of rifampin resistance detection using biochip and absolute concentration methods were congruent in 86% cases, and were different when analyzed samples consisted of the susceptible and resistant strains of M. tuberculosis mixture. The most frequent mutations in the rpoB gene were S531 (76.2%), H526 (7%), D516 (5.6%), and L511 (5.6%). In 94% of rifampin resistant strains, there was also resistance to isoniazid. Therefore, in Siberia the rifampin resistance is the reliable marker for MDR strains of M. tuberculosis, and biochips can be used also for their detection. To hybridize with biochip the fluorescent-labeled single-stranded DNAs were routinely synthesized by two PCR, and intermediary product after the first PCR should be transferred into another tube. The last stage included high risk of cross-contamination. To exclude the risk, primer concentrations and temperature-time profile of PCR reactions were improved, and both PCR were combined in one tube. The two methods were congruent in 100%. The one tube method would be especially attractive for the routine PCR laboratory.     

中国耐多药结核分枝杆菌临床分离株rpoB基因突变特点

为阐明中国耐多药结核分枝杆菌临床分离株rpoB基因的突变特征,对86株结核分枝杆菌临床分离株rpoB基因两个区域,包括81个碱基利福平抗药性决定区(rifampin resistance determining region,RRDR)和V176F区进行序列测定。其中72株耐多药分离株中的65株rpoB基因的RRDR区存在22种不同类型突变、21种点突变和一个插入突变。最常见的突变部位分别位于密码子531(41％)、526(40％)和516(4％),10％耐药分离株未检测到突变。鉴定了RRDR内6个新的等位基因,以及RRDR外部区域5个新的突变。所有分离菌株V176均无突变。     

Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods

Mutations in the rpoB locus confer conformational changes leading to defective binding of rifampin (RIF) to rpoB and consequently resistance in Mycobacterium tuberculosis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) was established as a rapid screening test for the detection of mutations in the rpoB gene, and direct sequencing has been unambiguously applied to characterize mutations. A total of 37 of Iranian isolates of M. tuberculosis, 16 sensitive and 21 resistant to RIF, were used in this study. A 193-bp region of the rpoB gene was amplified and PCR-SSCP patterns were determined by electrophoresis in 10% acrylamide gel and silver staining. Also, 21 samples of 193-bp rpoB amplicons with different PCR-SSCP patterns from RIFr and 10 from RIFs were sequenced. Seven distinguishable PCR-SSCP patterns were recognized in the 21 Iranian RIFr strains, while 15 out of 16 RIFs isolates demonstrated PCR-SSCP banding patterns similar to that of sensitive standard strain H37Rv. However one of the sensitive isolates demonstrated a different pattern. There were seen six different mutations in the amplified region of rpoB gene: codon 516(GAC/GTC), 523(GGG/GGT), 526(CAC/TAC), 531(TCG/TTG), 511(CTG/TTG), and 512(AGC/TCG). This study demonstrated the high specificity (93.8%) and sensitivity (95.2%) of PCR-SSCP method for detection of mutation in rpoB gene; 85.7% of RIFr strains showed a single mutation and 14.3% had no mutations. Three strains showed mutations caused polymorphism. Our data support the common notion that rifampin resistance genotypes are generally present mutations in codons 531 and 526, most frequently found in M. tuberculosis populations regardless of geographic origin.     

Characterization of rpoB mutations associated with rifampin resistance in Mycobacterium tuberculosis from eastern China

Aims:  The aim of this study was to investigate the features of rpoB gene mutations associated with Rifampin (RIF) resistance in Mycobacterium tuberculosis ( M. tuberculosis ) in eastern China.
Methods and Results:  The mutations of rpoB gene in 56 clinical isolates of M. tuberculosis resisted to one to four first-line drugs (rifampin, isonicotinyl hydrazide, ethambutol and streptomycin) were analysed by polymerase chain reaction single strand conformation polymorphism analysis (PCR-SSCP) and DNA sequencing. The results of PCR-SSCP showed 52 isolates were positive (existing rpoB mutation) including 47 isolates resisted to RIF. Subsequent results of DNA sequencing showed that 54 isolates had rpoB gene mutation including 49 isolates resisted to RIF. The most frequently mutated sites were at codons 526 (73·2%), 513 (10·7%) and 531 (3·5%).
Conclusions:  The rpoB codon 526 was the most frequently mutated site of RIF-resistant M. tuberculosis strains in eastern China and its frequency is significantly higher ( P  < 0·0001) compared with that in other areas of China and in other geographic regions worldwide.
Significance and Impact of the Study:  Our results reveal that geographic variation is responsible for rpoB mutations in M. tuberculosis and the resulting information will be helpful to improve a novel rapid molecular drug resistance screening approach for MDR TB.     

Antimicrobial susceptibility determined by the E test, Löwenstein-Jensen proportion, and DNA sequencing methods among Mycobacterium tuberculosis isolates discrepancies, preliminary results

Mycobacterium tuberculosis strains resistant to streptomycin (SM), isoniazid (INH), and/or rifampin (RIF) as determined by the conventional L?wenstein-Jensen proportion method (LJPM) were compared with the E test, a minimum inhibitory concentration susceptibility method. Discrepant isolates were further evaluated by BACTEC and by DNA sequence analyses for mutations in genes most often associated with resistance to these drugs (rpsL, katG, inhA, and rpoB). Preliminary discordant E test results were seen in 75% of isolates resistant to SM and in 11% to INH. Discordance improved for these two drugs (63%) for SM and none for INH when isolates were re-tested but worsened for RIF (30%). Despite good agreement between phenotypic results and sequencing analyses, wild type profiles were detected on resistant strains mainly for SM and INH. It should be aware that susceptible isolates according to molecular methods might contain other mechanisms of resistance. Although reproducibility of the LJPM susceptibility method has been established, variable E test results for some M. tuberculosis isolates poses questions regarding its reproducibility particularly the impact of E test performance which may vary among laboratories despite adherence to recommended protocols. Further studies must be done to enlarge the evaluated samples and looked possible mutations outside of the hot spot sequenced gene among discrepant strains.     

Simultaneous identification of rifampin-resistant Mycobacterium tuberculosis and nontuberculous mycobacteria by polymerase chain reaction-single strand conformation polymorphism and sequence analysis of the RNA polymerase gene (rpoB)

Interspecies variations and mutations associated with rifampin resistance in rpoB of Mycobacterium allow for the simultaneous identification of rifampin-resistant Mycobacterium tuberculosis and nontuberculous mycobacteria by PCR-SSCP analysis and PCR- sequencing. One hundred and ten strains of rifampin-susceptible M. tuberculosis, 14 strains of rifampin-resistant M. tuberculosis, and four strains of the M. avium complex were easily identified by PCR-SSCP. Of another seven strains, which showed unique SSCP patterns, three were identified as rifampin-resistant M. tuberculosis and four as M. terrae complex by subsequent sequence analysis of their rpoB DNAs (306 bp). These results were concordant with those obtained by susceptibility testing, biochemical identification, and 16S rDNA sequencing.     

Evaluation of reasons of the MDR M. tuberculosis strains dissemination by analysis of the rifampicin and/or isoniazid resistant isolates

During the last years in Novosibirsk region of Russia the rate of TB patients infected by MDR strains of M. tuberculosis has been constantly increasing. This increase may occur as a result of the spontaneously mutated mycobacterium selection during treatment of patients or as a result of primary infection by the resistant M. tuberculosis, or also, as a result of both reasons in combination. If the main reason of MDR strain dissemination is selection of resistant bacterium during patient treatment, the equal apportionment of the dominated mutation into the mycobacterium genotypes would be observed. If the main reason is the primary infection by resistant M. tuberculosis, the unequal apportionment would be revealed. For deeper understanding of the main reasons of the fast MDR strains spreading in the region, the distribution of the main mutations over genotypes of strains in Novosibirsk (170 isolates) and Tomsk prison (51 isolates) was investigated. Mutations in rpoB gene associated with the rifampicin resistance and in katG (isoniazid resistance) were detected by biochips. M. tuberculosis genotypings were carried out by IS6110 PCR typing or MIRU typing, in the last method the twelve loci (MIRU 2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39, 40) have been used. The most frequent mutation in the rpoB gene was Ser531-->Leu (60-70% of the rifampicin resistant strains) and Ser315-->Thr in gene katG (80% of the isoniazid resistant M. tuberculosis). Both in Novosibirsk and in Tomsk prison the rates of clustered cases transmissions were high (69 and 63% respectively). Analysis of the distribution of the dominated mutations Ser531-->Leu (rpoB) and Ser315-->Thr (katG) revealed that all of them were detected in each clusters, but in Novosibirsk there were only two clusters, in which the percentage of strains, containing mutation Ser531-->Leu (rpoB) were higher (85.7% and 77.7% respectively, P < 0.05), then in others. Among the Tomsk prison's clusters it was revealed one in which the proportion of the Ser3 15-->Thr mutation in katGwas higher (96.4%, P < 0.05). The nonuniform distribution of the dominated mutations highlighted that the epidemic spread of drug-resistant strains of M. tuberculosis in region resulted from the selection of them during patient treatment and the subsequent transmission by TB patients.     

结核耐药基因的基因芯片检测及临床应用

目的:采用基因芯片技术对结核分枝杆菌中常见耐药基因rpoB、katG及inhA进行检测,以了解结核分枝杆菌的耐药情况,及基因芯片技术检测结核菌耐药基因的临床应用价值。方法:收集40例涂片抗酸染色阳性并经分枝杆菌菌种鉴定芯片鉴定为结核的样本进行结核耐药基因检测。结果:40例样本中,14例无法判读结果,占35%,检出26例,检出率为65%。其中,无突变的野生型21例,占52.5%;突变型5例,总突变率为12.5%;3例rpoB基因的531点单独突变(TCG→TTG),突变率为7.5%;2例katG基因的315点单独突变(AGC→ACC),突变率为5%。结论:结核耐药基因芯片试剂盒检测结核菌耐药基因时针对单个菌落,用痰样本直接检测耐药基因虽能简便快速地了解结核分枝杆菌的耐药情况,但会出现一些无法判读的结果,原因须进一步探讨。