用噬菌体检测技术检测结核分支杆菌的临床应用评价

目的评价噬菌体检测技术检测临床标本中结核分支杆菌的优缺点.方法对52名活动性肺结核患者的痰标本分别用涂片镜检法、噬菌体法、聚合酶链反应(PCR)法进行结核分支杆菌检测,同时选取15例正常人痰标本作为对照组.结果 3种方法检测15例对照组标本均无1例阳性.但研究组噬菌体法阳性标本48例,镜检法阳性标本38例,PCR阳性标本50例,其中痰涂片镜检阳性者,噬菌体法和PCR法均为阳性;而50例PCR法阳性者包含了48例噬菌体法阳性者,另有2例仅PCR法阳性.结论噬菌体法检测结核分支杆菌立意新颖,准确可靠.其对结核分支杆菌的检出率大大高于涂片镜检法,同时又不需要特殊的实验室和实验设备,值得临床检验科推广使用.     

TDI-FP技术检测耐乙胺丁醇结核分支杆菌embB306点突变

目的 应用TDI-FP技术分析结核分支杆菌embB 306点突变与乙胺丁醇耐药性的关系，并建立一种准确、快速的诊断结核分支杆菌乙胺丁醇耐药的新方法。方法 对临床82例耐多药结核病患者所感染的结核分支杆菌菌株进行常规培养和药敏实验；提取结核分支杆菌DNA，并PCR扩增205bp的embB基因片段，消化降解掉PCR产物中残余的dNTPs和引物，进行模板指导的荧光标记终止碱基掺入反应；应用Victor2测定反应产物的荧光偏振值，分析所有样品的embB基因306位点的基因型；对分析结果进行测序验证。结果 5例乙胺丁醇高度耐药患者中有3例所感染的结核分支杆菌发生embB 306的ATG→GTG突变；47例乙胺丁醇低度耐药患者中有15例为embB 306突变和未突变的混合感染；30例乙胺丁醇敏感患者的结核分支杆菌未发现embB 306突变。测序结果与实验相符合。结论 embB 306突变与结核分支杆菌对乙胺丁醇产生耐药性密切有关；应用TDI-FP技术可以准确、快速简便检测embB 306突变，在结核分支杆菌耐乙胺丁醇的临床诊断中具有潜在的应用前景。     

基因芯片技术检测结核分支杆菌耐利福平基因突变的研究

目的建立基因芯片检测结核分支杆菌耐利福平基因突变，结合基因测序和常规药物敏感性试验进行比较，探讨其实用价值。方法根据结核菌rpoβ基因突变特点，设计不同的寡核苷酸探针，点样制备可检测rpoβ基因突变的芯片，检测利福平（Rifompicin，RFP）株的rpoβ变异情况。结果137株临床分离结核菌，49株耐RFP，占35．8％，平均MIC为179．6±135．0ug／mL。Rpop突变率为95．7％（47／49），主要为点突变。突变点依次为531 Ser（TCG）→Leu（TTG）（40．8％）、531 Ser（TCG）→Trp（TGG）（12．2％）、526 His（CAC）→Tyr（TAC）（6．1％）、526His（CAC）→Asp（GAC）（6．1％）。526 His（CAC）→Asn（AAC）（6．1％）、526 His（CAC）→Pro（CCC）（8．2％）、516Asp（GAC）→Val（GTC）（12．2％）、533 Leu（CTG）→Arg（CGG）（4．1％）和513 GIn（CAA）→Pro（CCA）（2．1％）。结论本研究建立的检测结核菌rpoβ基因突变的基因芯片技术敏感性高，特异性强，可应用于结核菌耐RFP基因的检测。     

PCR技术在结核分支杆菌诊断及耐药性检测中的应用

对肺结核及肺外结核的早期诊断一直是临床医学的一个挑战;痰涂片检查简单易行但检出率低;结核菌培养是诊断的金标准但费时且菌量较少时出现假阴性是其主要缺陷.近年来结核杆菌耐药株的增多使问题更趋复杂.聚合酶链反应(PCR)检测技术,以灵敏度高、特异性强、简便、快速等优点,在结核分支杆菌的快速诊断及耐药性检测方面发挥了重要作用.     

均相荧光探针PCR技术检测结核分支杆菌的评价

近年来,用PCR方法（凝胶电泳后,经溴化乙锭染色）检测结核分支杆菌,为结核病的诊断提供了简便,快速的方法。但是,在实际工作中,由于某些干扰因素的存在,PCR电泳图谱存在多形性,给结果观察和判断带来一定困难,造成一定的假阳性及假阴性的存在[1]。应用均相荧光探针PCR对病原作的DNA进行检测是一项最新的技术。我们用这种方法与PCR（电泳EB染色）,涂片,培养进行了比较,得到了较满意的效果。均相荧光深外Itll技术的原理是：在poB扩增系统中加入结合荧光标记发夹式寡接着酸探针,该探针的5’和3’端分别标记荧光染料和摔灭…     

DNA芯片法检测耐多药结核分支杆菌耐药性

目的 :应用新型DNA芯片快速检测结核分枝杆菌对异烟肼利福平的耐药性。方法 :2 0株耐INH、RFP结核分支杆菌临床分离株 ,PCR扩增并荧光标记包含结核分支杆菌katG、rpoB基因突变热点的片段 ,与芯片杂交。结果 :耐INH、RFP2 0株经DNA芯片检测 ,14株katG基因突变 ( 70 % )、17株rpoB基因突变 ( 85 % )。结论 :用DNA芯片检测结核分支杆菌株耐药性有较高的特异性和灵敏度 ,且快捷     

荧光定量PCR技术检测结核分支杆菌DNA的应用价值

目的评价荧光定量PCR技术检测临床标本中结核分支杆菌的应用价值。方法应用荧光定量PCR法及抗酸染色、结核杆菌培养法,对179例活动性肺结核患者晨痰、123例活动性肺结核患者外周血、34例结核性胸膜炎患者胸水、20例非结核性呼吸系统疾病患者的晨痰标本进行检测。结果179例活动性肺结核患者晨痰、123例活动性肺结核患者外周血、34例结核性胸膜炎患者胸水,涂片抗酸染色阳性率分别为20．67％（37／179）、0．00％、0．00％;细菌培养阳性率分别为41．34％（74／179）、0．00％、2．94％（1／34）;荧光定量PCR技术阳性率分别为64．25％（115／179）、38．21％（47／123）、44．12％（15／34）。3种标本荧光定量PCR技术阳性率高于抗酸染色和结核杆菌培养,经统计学处理发现,差异有统计学意义。用荧光定量PCR技术检测临床标本特异性为95．00％。结论荧光定量PCR技术将PCR扩增、荧光探针杂交及检测一体化,在单一管内完成,具有简便、快速、防污染、敏感性及特异性高等优点,是结核病诊断的有效方法之一。     

PCR-SSCP法检测耐多药结核分支杆菌耐药性

我国是结核病疫情最严重的国家之一，其主要原因是耐药和耐多药结核病的广泛分布和迅速传播。异烟肼（INH）和利福平（RFP）均是抗结核的一线药物，对于其作用机理及结核杆菌的耐药机制的研究日益受到重视。据报道认为结核分支杆菌耐与RNA聚合酶的8亚基的编码基因rpoB的突变有关；耐异烟肼的产生是过氧化氢酶一过氧化物酶的编码基因KatG基因的缺失和突变有关，也inhA基因突变也有相关性。我们采用PCR—SSCP银染法直接检测临床分离株中结核分支杆菌rpoB基因和KatG基因突变，现将结果报告如下。     

耐异烟肼结核分支杆菌分子生物学研究进展

近年来结核病再度流行,耐药结核菌株的出现是重要原因之一。异烟肼作为治疗结核病最主要的一线药物,其耐药率显著上升,我国异烟肼耐药率已达20％以上。结核分支杆菌对异烟肼的耐药机制较为复杂,涉及多个分子靶位,近年来在结核病研究中倍受关注,国内外已有这方面的研究报道。本文对近年来耐异烟肼结核分支杆菌的分子生物学研究进展作一综述。     

结核分支杆菌耐链霉素耐药基因的检测

目的：探讨结核分支杆菌对链霉素(SM)的耐药分子机制,评估应用聚合酶链反应－单链构象多态性（PCR－SSCP）技术检测rpsL基因突变在结核分支杆菌对SM耐药性测定中的应用价值。方法：采用PCR－SSCP方法对71株结核分支杆菌临床分离株（其中药物敏感株35株,耐SM或含耐SM耐多药株36株）和30例耐SM肺结核病人痰标本的rpsL基因突变进行检测。结果：以结核分支杆菌H37RV为对照,35株药物敏感株的rpsL基因PCR扩增产物267bp片段SSCP图谱正常;36株耐SM分离株中,26株rpsL基因SSCP图谱异常,其突变率为72．2％。30例耐SM肺结核病人痰标本有22例PCR扩增阳性,14例SSCP图谱与H37RV株有差异,rpsL突变率为63．6％。结论：rpsL基因突变是SM耐药性的重要分子机制,PCR－SSCP技术可以快速、准确地检测结核分支杆菌rpsL基因突变,该技术有望直接用于临床标本耐药性检测。     

聚合酶链反应-测序技术快速检测耐异烟肼结核分枝杆菌KatG基因突变的研究

目的 应用PCR-DNA测序技术快速检测耐异烟肼结核分枝杆菌分离株KatG基因突变,评价其在检测结核分枝杆菌异烟肼耐药性方面的应用价值.方法 47株耐异烟肼结核分枝杆菌临床分离株及30株结核分枝杆菌敏感分离株用PCR-DNA测序技术检测KatG基因突变.结果 47株耐异烟肼结核分枝杆菌分离株中,有31株KatG基因检出有突变,突变检出率为66.0%(31/47);30株结核分枝杆菌敏感株检出1株KatG基因突变.结论 PCR-DNA测序技术方法敏感、准确、特异,可快速检测结核分枝杆菌KatG耐药基因突变,有利于耐异烟肼结核分枝杆菌耐药性的快速检测.     

聚合酶链反应-测序技术快速检测耐异烟肼结核分枝杆菌KatG基因突变的研究

目的应用PCR-DNA测序技术快速检测耐异烟肼结核分枝杆菌分离株KatG基因突变,评价其在检测结核分枝杆菌异烟肼耐药性方面的应用价值。方法47株耐异烟肼结核分枝杆菌临床分离株及30株结核分枝杆菌敏感分离株用PCR-DNA测序技术检测KatG基因突变。结果47株耐异烟肼结核分枝杆菌分离株中,有31株KatG基因检出有突变,突变检出率为66.0%(31/47);30株结核分枝杆菌敏感株检出1株KatG基因突变。结论PCR-DNA测序技术方法敏感、准确、特异,可快速检测结核分枝杆菌KatG耐药基因突变,有利于耐异烟肼结核分枝杆菌耐药性的快速检测。     

Differentiation of Mycobacterium tuberculosis and Mycobacterium bovis BCG by a polymerase chain reaction assay

Mycobacterium tuberculosis and Mycobacterium bovis are closely related species which carry different numbers of the repetitive DNA element IS6110. A polymerase chain reaction assay was developed to assess the copy number of IS6110 in a strain and thereby differentiate these two important human pathogens.     

Mycobacterium tuberculosis detection by polymerase chain reaction and identification of M. tuberculosis strain

A rapid multiprimer PCR method for detection of Mycobacterium tuberculosis complex (MTC) and simultaneous identification of M. tuberculosis in clinical samples has been developed. The method is based on simultaneous amplification of two targets: a 401 bp region from the mtp40 species-specific gene sequence of M. tuberculosis and a 544 bp fragment from the RD1 genome region which is specific for MTC but absent in BCG strains. Polymerase inhibitors in this study were detected by internal control in each test. Detection sensitivity was 25 copies of M. tuberculosis genomic DNA. Seven methods for isolation of mycobacterial DNA were compared and the technique with chloroform extraction was selected as the most efficient. The proposed method was used for analysis of 37 clinical samples and the results were compared with the results of culturing, acid-fast bacilli staining, and clinical diagnosis. The method proved to be sufficiently sensitive and specific for detection of mycobacterial DNA. Moreover, in countries with only two main pathogenic species of MTC circulating (M. tuberculosis and M. bovis) this method can be used for differentiation of these two species.     

Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis

The molecular basis for isoniazid resistance in Mycobacterium tuberculosis is complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance. We present the first large-scale analysis of 240 alleles previously associated with isoniazid resistance in a diverse set of 608 isoniazid-susceptible and 403 isoniazid-resistant clinical M. tuberculosis isolates. We detected 12 mutant alleles in isoniazid-susceptible isolates, suggesting that these alleles are not involved in isoniazid resistance. However, mutations in katG, ahpC, and inhA were strongly associated with isoniazid resistance, while kasA mutations were associated with isoniazid susceptibility. Remarkably, the distribution of isoniazid resistance-associated mutations was different in isoniazid-monoresistant isolates from that in multidrug-resistant isolates, with significantly fewer isoniazid resistance mutations in the isoniazid-monoresistant group. Mutations in katG315 were significantly more common in the multidrug-resistant isolates. Conversely, mutations in the inhA promoter were significantly more common in isoniazid-monoresistant isolates. We tested for interactions among mutations and resistance to different drugs. Mutations in katG, ahpC, and inhA were associated with rifampin resistance, but only katG315 mutations were associated with ethambutol resistance. There was also a significant inverse association between katG315 mutations and mutations in ahpC or inhA and between mutations in kasA and mutations in ahpC. Our results suggest that isoniazid resistance and the evolution of multidrug-resistant strains are complex dynamic processes that may be influenced by interactions between genes and drug-resistant phenotypes.     

焦磷酸测序技术检测耐多药结核分枝杆菌

目的利用焦磷酸测序技术,建立结核分枝杆菌菌株耐多药检测方法,并与Bactec MGIT 960药敏法进行比较。方法设计rpoB、katG基因PCR扩增引物和焦磷酸测序引物,建立结核分枝杆菌菌株焦磷酸测序检测方法。利用新确立的焦磷酸测序法检测202株临床分离的耐多药结核分枝杆菌的利福平、异烟肼耐药性。结果与BACTEC MGIT 960法检测药物耐药性结果比较,焦磷酸测序检测多耐药结核分枝杆菌临床分离株的敏感性与特异性为72.8%[95%可信区间(confidence in-terval,CI):66.3%~78.4%]及90.0%(95%CI:80.1%~95.1%)。结论新确立的焦磷酸测序技术可快速检测结核分枝杆菌对利福平、异烟肼的耐药性,其可作为耐多药结核分枝杆菌药敏有效的检测工具。     

Rapid detection of rifampin-resistant Mycobacterium tuberculosis directly from stained sputum smears using single-tube nested polymerase chain reaction deoxyribonucleic acid sequencing

Microscopy is the mainstay of laboratory diagnosis of tuberculosis especially in resource poor countries. The World Health Organization has also recommended microscopy as the mainstay of diagnosis for directly observed treatment, short course. Using DNA extracts from Ziehl-Neelsen (ZN)-stained sputum smears, a single-tube nested polymerase chain reaction was optimized to confirm Mycobacterium tuberculosis complex and detect rifampin (RIF) resistance by sequencing, using a combination of novel (rpoB47 and rpoB158) and previously described (rpoB105 and rpoB293) primers. Carryover DNA was strictly monitored using several negative controls, and inhibition was ruled out by spiked controls. No such target was detected from negative controls and purified genomic DNA from other nontubercular mycobacteria. Resistance could be detected in 91.1% (51/56) slides. The results obtained were concordant with the 1% proportion method and DNA sequencing performed on culture isolates. Our results demonstrate that the method is suitable for rapid detection of susceptibility to RIF in acid-fast bacillus-positive ZN-stained slides obtained from patients suspected of harboring drug-resistant M. tuberculosis.