结核分枝杆菌三种耐药基因的检测方法

建立3种结核分枝杆菌耐药基因的检测方法,探讨耐药基因突变与耐药性的关系。将58株临床分离株均做聚合酶链反应-单链构象多态性分析(PCR—SSCP)和传统药物敏感试验。结果表明,结核分枝杆菌耐药基因突变与耐药水平有密切联系,绝大多数结核分枝杆菌耐药基因突变发生在高耐药株,少部分在低耐药株发生基因突变。     

结核分枝杆菌临床株4种耐药基因的检测与分析

结核分枝杆菌Mycobacterium tuberculsis(M.t)4种耐药基因的研究,了解耐药基因突变情况和耐药水平的关系。108例临床痰标本临床分离株均做传统梯度药敏试验和聚合酶链反应多态-单链构象多态性（PCR-SSCP）试验。结果表明耐SM（rpsL）REP(rpoB）INH(katG）EMB（embB）基因突变率分别为78.5%,68.2%,70.5%,48.6%。其中,上海高耐药株基因突变率分别为86.5%,89.3%,84.3%,35.3%。低耐药株分别为28.5%,16.5%,7.1%。EMB在低耐药区无基因突变。M.t的4种耐药基因联合检测的分析,在国内外很少报道。部分M.t的耐药由耐药基因突变所致,M.t耐药基因突变与耐药水平密切相关,且M.t基因突变绝大多数发生在高耐药区中,也有少部分在低耐区株中发生。     

应用4种方法检测结核分枝杆菌临床分离株对链霉素的耐受性

通过DNA测序、SSCP、RFLP和反向斑点杂交技术分析167株结核分枝杆菌临床分离株的耐药基因型,评价结核分枝杆菌rpsL或rrs基因突变与链霉素（SM）耐受性之间的关系,比较4种分子方法检测SM耐受性的临床价值。98株耐SM分离株中,78株（79．6％）rpsL 43位或88位密码子错义突变导致赖氨酸置换为精氨酸,6株（6．1％）rrs 513位碱基A突变为C或T或516位C突变为T,14株（14．3％）未发现突变;69株SM敏感的分离株未发现这两个基因突变。应用SSCP、RFLP和RDBH方法分析上述突变和野生序列的结果与DNA测序完全一致,RDBH方法可从98株耐SM分离株中正确鉴定出84株（85．7％）分离株的5种突变基因型。结果表明,应用分子技术分析rpsL和rrs基因突变可快速检测大多数结核分枝杆菌对SM的耐受性,反向斑点杂交方法是一个快速、简便和可靠地检测药物耐受性的分子方法。     

Detection of Streptomycin Resistance in Mycobacterium tuberculosis Clinical Isolates Using Four Molecular Methods in China

To evaluate the relationship between mutations in rpsL or rrs genes and streptomycin (SM) resistance, we compared four molecular methods for their clinical value in the detection of SM resistance. Genotypic analysis of SM resistance in 167 M. tuberculosis clinical strains isolated from Chinese patients was performed by direct DNA sequencing, SSCP, RFLP, and reverse dot-blot hybridization (RDBH) assays. Of the 98 SM-resistant isolates, 78 (79.6%) had missense mutations in codon 43 or 88 of rpsL resulting in a Lys to Arg substitution, 6 (6.1%) had mutations of the rrs gene at positions 513 A to C or T or 516 C to T, and 14 (14.3%) had the wild-type sequence. None of the 69 SM-susceptible isolates examined had alterations in rpsL or rrs. The results of the SSCP, RFLP, and RDBH analyses for these mutations and wild-type sequences were completely consistent with DNA sequencing data. Five distinct single-nucleotide substitutions in codon 43 or 88 of rpsL gene or in position 513 or 516 of rrs gene were correctly identified in 84 of 98 (85.7%) phenotypically SM-resistant isolates by RDBH assay. Molecular analyses of the rpsL and rrs genes are useful for rapid prediction of SM resistance in most clinical strains of M. tuberculosis. Reverse dot-blot hybridization assay is a rapid, simple, and reliable method for the detection of drug resistance.     

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.     

Detecting drug-resistant tuberculosis: the importance of rapid testing

Despite numerous intervention strategies, including the direct observed short-course treatment strategy and improved diagnostic methods, the incidence of multidrug-resistant and extensively drug-resistant tuberculosis (TB) continues to rise globally. Many treatment policies are based on the model that acquisition of drug resistance in already infected individuals drives the drug-resistant TB epidemic, hence the focus on drug-resistance testing of retreatment cases. However, molecular epidemiology and mathematical modeling suggest that the majority of multidrug-resistant TB cases are due to ongoing transmission of multidrug-resistant strains. This is most likely the result of diagnostic delay, thereby emphasizing the need for rapid diagnostics and comprehensive contact tracing, as well as active case finding. Current diagnosis of TB in low-income, high-burden regions relies on smear microscopy and clinical signs and symptoms. However, this smear-centered approach has many pitfalls, including low sensitivity in HIV patients and children, the inability of smear to reveal drug-resistance patterns, and the need for sampling on consecutive days. In order to address these limitations, efforts have been made to expand access to Mycobacterium tuberculosis culture and drug susceptibility testing. However, the slow growth rate of the causative agent, M. tuberculosis, contributes to significant diagnostic delay. Molecular-based diagnostic methods, targeting mutations that are known to confirm drug resistance, are capable of significantly reducing diagnostic delay. Two such methods, the line-probe assay and the real-time PCR-based Xpert? MTB/RIF assay, have been described. The latter test shows particular promise for smear-negative and extrapulmonary specimens. This may prove especially useful in settings where co-infection rates with HIV are high. However, since most research focuses on the performance of both of these assays, further investigations need to be done regarding the impact of the routine implementation of these assays on TB control programs and the cost effectiveness thereof.     

结核分枝杆菌katG基因突变的研究

探讨编码过氧化氢-过氧化物酶的katG基因突变与结核分枝杆菌异烟肼(INH)耐药性的相关关系。根据结核分枝杆菌GenBank中的katG序列,自行设计特异性寡聚核苷酸引物,采用聚合酶链反应-单链构象多态性(PCR-SSCP)分析和直接测序法(DS)分析结核分枝杆菌中katG基因突变情况。以HR37Rv标准株为对照。所有23株敏感菌均未有SSCP结果异常;35株耐药菌中,有2株(5．7％)katG基因扩增阴性,且发生在高度耐药菌中。进一步分析发现,SSCP法突变检出23株(65．7％),测序法突变检出24株(68．6％),符合率为95．8％(23／24)。参照测序法对耐药菌突变序列的分析结果,PCR—SSCP敏感、特异,可快速检测结核分枝杆菌katG耐药基因突变,有利于耐药结核分枝杆菌耐药性的快速检测。     

Transmission of synovial sarcoma from a single multi-organ donor to three transplant recipients: case report

Quick and reliable testing of EGFR and KRAS is needed in non-small cell lung cancer (NSCLC) to ensure optimal decision-making for targeted therapy. The Idylla™ platform was designed for Formalin-Fixed Paraffin-Embedded (FFPE) tissue sections but recently several studies were published that evaluated its potential for cytological specimens. This study aimed to validate the Idylla™ platform for the detection of EGFR/KRAS mutations in cytological NSCLC samples prepared as cytoblocks using AGAR and paraffin embedding. The KRAS Idylla™ test were performed on 11 specimens with a known KRAS mutation. The EGFR Idylla™ test was performed on 18 specimens with a known primary EGFR mutation and 7 specimens with a primary EGFR-EGFR T790M resistance mutation combination. Concordant KRAS and primary EGFR mutations were detected for both KRAS and primary EGFR mutations. Samples with a total CQ value of < 26 could be considered negative. Samples with a total CQ value of > 26 could not be assessed (probability of false-negative). In specimens with a primary EGFR-EGFR T790M resistance mutation combination, 5/7 cases were not concordant. Our results confirm the conclusion of recent reports that the Idylla™EGFR assay is not suitable in a resistance to EGFR TKI setting, also not in our cytological NSCLC samples prepared as cytoblocks using AGAR and paraffin embedding. KRAS and primary EGFR mutations were detected using the Idylla™ assays in virtually all cytological NSCLC samples. This analysis was rapid and time-saving compared to other mutation detection assays and may be useful if the amount of material is insufficient to perform a full set of molecular tests.     

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。     

A new Multi-PCR-SSCP assay for simultaneous detection of isoniazid and rifampin resistance in Mycobacterium tuberculosis

Prompt detection of drug resistance in Mycobacterium tuberculosis is essential for effective control of tuberculosis (TB). We developed a Multi-PCR-SSCP method that detects more than 80% commonly observed isoniazid (INH) and rifampin (RIF) resistance M. tuberculosis in a single assay. The usefulness of the newly developed method was evaluated with 116 clinical isolates of M. tuberculosis. Distinct SSCP patterns were observed for different mutations and the correlation between Multi-PCR-SSCP results and DNA sequencing data was strong. Using the culture-based phenotypic drug susceptibility testing as a reference, the sensitivity of the newly developed Multi-PCR-SSCP assay was determined to be 80% and 81.8% for INH and RIF, respectively. The specificity of the assay was 100% and 92%, for INH and RIF, respectively. Multi-PCR-SSCP provides a rapid and potentially more cost-effective method of detecting multidrug-resistant TB.     

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.     

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.     

Single strand conformation polymorphism is a sensitive method for screening nucleotide variations in Mycosphaerella graminicola

Single Strand Conformation Polymorphism (SSCP) and sequencing were performed in order to assess molecular polymorphism of mating type sequences in the heterothallic ascomycete Mycosphaerella graminicola, the causal agent of Septoria tritici blotch of wheat. The screening was undertaken on mat1-1 and mat1-2 partial sequences of 341 and 657 bp, respectively, amplified with multiplex PCR from 510 French single-conidial strains plus the two reference isolates IPO323 and IPO94269 from The Netherlands. After restriction with Taq1 in order to reduce the fragment sizes, all digested amplicons were subjected to SSCP. Sequencing was then performed when a SSCP pattern deviates from the most frequently occurring profile. Among the assessed strains, 228 ones plus IPO323 were MAT1-1 and 282 ones plus IPO94269 were MAT1-2. Among the MAT1-1 strains, only a single one exhibited a SSCP profile distinct to the other MAT1-1 strains, whereas 10 MAT1-2 strains (among which 2 and 4 with same profiles, respectively) showed a SSCP profile differing to the other MAT1-2 strains. Sequencing revealed that all polymorphisms observed on SSCP gels were single nucleotide variations and all strains displaying the same SSCP profiles showed identical nucleotide sequences. Among the seven disclosed nucleotide variations, only two were non-synonymous and both were non-conservative. This study reports a high sensitivity of SSCP allowing detection of single point mutations in M. graminicola, shows a conservation of mating type idiomorphs in the fungus at both sequence and population scales, but also suggests a difference in polymorphism level between the two mating type sequences.     

Site-directed mutagenesis of the katG gene of Mycobacterium tuberculosis: effects on catalase–peroxidase activities and isoniazid resistance

Recent studies examining the molecular mechanisms of isoniazid (INH) resistance in Mycobacterium tuberculosis have demonstrated that a significant percentage of drug-resistant strains are mutated in the katG gene which encodes a catalase–peroxidase, and the majority of these alterations are missense mutations which result in the substitution of a single amino acid. In previous reports, residues which may be critical for enzymatic activity and the drug-resistant phenotype have been identified by evaluating INH-resistant clinical isolates and in vitro mutants. In this study, site-directed mutagenesis techniques were utilized to alter the wild-type katG gene from M. tuberculosis at 13 of these codons. The effects of these mutations were determined using complementation assays in katG -defective, INH-resistant strains of Mycobacterium smegmatis and Mycobacterium bovis BCG. This mutational analysis revealed that point mutations in the katG gene at nine of the 13 codons can cause drug resistance, and that enzymatic activity and resistance to INH are inversely related. In addition, mutations in the mycobacterial catalase–peroxidase which reduce catalase activity also decrease peroxidase activity.     

结核分枝杆菌耐链霉素和乙胺丁醇分子机制的研究

目的:研究结核分枝杆菌耐链霉素和乙胺丁醇的rpsL和emb B基因突变情况,探讨耐药基因突变与耐药性的关系。方法:通过传统药敏实验和聚合酶链反应(PCR)--单链构象多态性(SSCP)技术初步鉴定62株临床分离株的药敏和rps L、emb B基因。结果:与结核菌标准株H37Rv对照,分析30例TB菌耐链霉素(SM)的rps L基因,发现其突变率为70.0%(21/30),分析29例耐乙胺丁醇(EMB)的emb B基因,该基因的突变率为65.5%(19/29)。结论:部分结核分枝杆菌耐SM和EMB是由于其rps L、emb B基因突变所致,PCR-SSCP银染技术可能成为测定部分结核分枝杆菌耐药的简便、快速的方法。     

Technology platforms for molecular diagnosis of cystic fibrosis

Cystic fibrosis (CF) is one of the most common recessive genetic diseases in North America. So far, 1200 mutations causing CF have been identified. Several techniques such as allele specific oligonucleotide (ASO) dot-blot, reverse dot-blot, amplification refractory mutation (ARMS), and an oligo-ligation assay, are available to detect the most common mutations. However, detecting compound heterozygotes between DeltaF508, the most common disease causing mutation, and other mutations which are rare is difficult as some mutations are common only to particular ethnic groups. Therefore, new diagnostic tests such as restriction enzyme assays and single stranded conformational polymorphism (SSCP) have been designed to recognize rare and population-specific mutations. This review will describe the most commonly used CF mutation detecting diagnostic techniques, as well as novel assays and techniques currently in development that might be employed in future.     

Analysis of the genetic determinants of multidrug and extensive drug resistance in Mycobacterium tuberculosis with the use of an oligonucleotide microchip

Steadily growing resistance of the tuberculosis causative agent towards a broad spectrum of antituberculosis drugs calls for rapid and reliable methods for identifying the genetic determinants responsible for this resistance. In this study, we present a biochip-based method for simultaneous identification of mutations within rpoB gene associated with rifampin resistance, mutations in katG, inhA, ahpC genes responsible for isoniazid resistance, mutations within the regions of gyrA and gyrB genes leading to fluoroquinolones resistance, and mutations in the rrs gene and the eis promoter region associated with the resistance to kanamycin, capreomycin and amikacin. The oligonucleotide microchip, as the core element of this assay, provides simultaneous identification of 99 mutations in the format “one sample—one PCR—one microchip”, and it makes it possible to complete analysis of multidrug-resistant and extensively drug-resistant tuberculosis within a single day. The tests on 63 Mycobacterium tuberculosis clinical isolates with different resistance profiles using the developed approach allows us to reveal the spectrum of drug-resistance associated mutations, and to estimate the significance of the inclusion of extra genetic loci in the determination of M. tuberculosis drug resistance.     

Loss of a conserved 7-methylguanosine modification in 16S rRNA confers low-level streptomycin resistance in bacteria

Streptomycin has been an important drug for the treatment of tuberculosis since its discovery in 1944. But numerous strains of Mycobacterium tuberculosis, the bacterial pathogen that causes tuberculosis, are now streptomycin resistant. Although such resistance is often mediated by mutations within rrs, a 16S rRNA gene or rpsL, which encodes the ribosomal protein S12, these mutations are found in a limited proportion of clinically isolated streptomycin-resistant M. tuberculosis strains. Here we have succeeded in identifying a mutation that confers low-level streptomycin resistance to bacteria, including M. tuberculosis. We found that mutations within the gene gidB confer low-level streptomycin resistance and are an important cause of resistance found in 33% of resistant M. tuberculosis isolates. We further clarified that the gidB gene encodes a conserved 7-methylguanosine (m(7)G) methyltransferase specific for the 16S rRNA, apparently at position G527 located in the so-called 530 loop. Thus, we have identified gidB as a new streptomycin-resistance locus and uncovered a resistance mechanism that is mediated by loss of a conserved m(7)G modification in 16S rRNA. The clinical significance of M. tuberculosis gidB mutation also is noteworthy, as gidB mutations emerge spontaneously at a high frequency of 10(-6) and, once emerged, result in vigorous emergence of high-level streptomycin-resistant mutants at a frequency more than 2000 times greater than that seen in wild-type strains. Further studies on the precise function of GidB may provide a basis for developing strategies to suppress pathogenic bacteria, including M. tuberculosis.     

Direct rapid diagnosis of rifampicin-resistant M. tuberculosis infection in clinical samples by line probe assay (INNO LiPA Rif-TB)

M. tuberculosis is one of the leading causes of death worldwide and Multi Drug Resistant Tuberculosis (MDR-TB) is associated with a high case-fatality rate. Rapid identification of resistant strains is crucial to institute prompt appropriate therapy, and prevent the development of further resistance and spreading of MDR strains. The INNO-LiPA Rif. TB is a commercial reverse hybridisation line probe assay designed for rapid detection of rpoB gene mutations in clinical isolates. We applied this test directly to 44 smear-positive and 45 smear-negative clinical specimens collected from patients suspected of active TB. The capability of this technique to correctly identify local MDR-TB strains was tested on 50 MDR strains isolated in Italy. Results of the test were compared to conventional antibiogram performed on isolated strains. The concordance rate of the LiPA test results on clinical specimens with those obtained with "in vitro" sensitivity was 100%. These results show that the LiPA test can be useful in rapid detection and prompt management of tuberculosis when MDR disease is suspected.