结核分支杆菌利福平耐药基因突变的研究

目的了解我国结核分支杆菌耐利福平（ＲＦＰ）分离株ｒｐｏＢ基因突变情况，建立快速检测结核分支杆菌耐药基因型的分子药敏试验方法。方法通过聚合酶链反应－单链构象多态性（ＰＣＲ－ＳＳＣＰ）和ＰＣＲ－直接测序法（ＰＣＲ－ＤＳ）分析５０株结核分支杆菌临床分离株的ｒｐｏＢ基因。结果３株结核分支杆菌药物敏感株和２株非ＲＦＰ耐药株中，仅１株ｒｐｏＢＳＳＣＰ图谱异常的药物敏感株出现５３１位密码子ＴＣＧ→ＴＴＧ突变。４５株ＲＦＰ耐药株中，１０株ＳＳＣＰ和ＤＳ分析均未见ｒｐｏＢ突变，３５株ＳＳＣＰ和ＤＳ分析异常，其中１４株为５３１位密码子ＴＣＧ→ＴＴＧ或ＴＧＧ或ＴＡＣ突变，１４株为５２６位ＣＡＣ→ＴＡＣ或ＧＡＣ或ＣＣＣ或ＣＴＣ或ＧＴＣ突变，２株为５１６位ＧＡＣ→ＧＴＣ或ＴＡＣ突变，２株为５１６位和５２６位及５１５位和５１６位密码子双点突变，３株ｒｐｏＢ序列与结核分支杆菌不同。结论大多数结核分支杆菌耐ＲＦＰ是由于其ｒｐｏＢ基因突变所致，采用ＰＣＲ－ＳＳＣＰ和ＰＣＲ－ＤＳ方法可快速测定结核分支杆菌ＲＦＰ耐药基因型     

耐多药结核分支杆菌三种耐药基因检测

报道聚合酶链反应单链构象多态性（ＰＣＲＳＳＣＰ）方法检测耐多药结核分支杆菌ｒＰＯＢ、ｒＰＳＬ和ＫａｔＧ基因突变情况。材料和方法（１）菌株：结核分支杆菌Ｈ３７Ｒｖ参考菌株购自中国菌种保存中心；１０２株结核分支杆菌临床分离株取自本市肺结核患者，常规进...     

DNA指纹技术方法学的建立及其在结核菌株鉴定中的应用

目的探讨结核分支杆菌ＤＮＡ指纹技术的方法学及其在结核菌株水平鉴定中的应用。方法以结核分支杆菌染色体ＤＮＡ插入序列ＩＳ６ＩＩＯ为基础，根据不同来源的结核分支杆菌菌株ＤＮＡ有相异的ＩＳ６１１０拷贝数和分子量，应用增强化学发光标记技术对结核分支杆菌ＤＮＡ酶切产物进行杂交和检测。结果（１）不同来源的５０例结核病人临床分离株具有相异的ＤＮＡ指纹谱。（２）－病人肺部痰和膝关节脓培养阳性的结核菌株ＤＮＡ指纹图谱     

广东省肺结核临床分离菌株DNA指纹分析

目的 构建结核分支杆菌IS6110 DNA指纹图谱，从分子水平探讨广东省结核分支杆菌的特征。方法 参照Van Embden推荐的结核分支杆菌DNA指纹标准方法，构建标准菌株Mt14323和广东省的74株临床分离株的以RFLP为基础的结核分支杆菌的IS6110 DNA指纹图谱；经互联网与世界结核菌DNA指纹库进行相似性比较；应用Gel compar 4．1(Applied Maths，Kortrijk，Belgium)软件对上述菌株的指纹图谱进行聚类分析。结果 Mt14323标准菌株和74例临床分离菌株IS6110DNA指纹图谱结果与国外同类报道一致；其中24．3％(18／74)的结核菌株的IS6110 DNA指纹相似值在1-0．65之间，鉴定结果为它们均是北京家族结核分支杆菌；IS61100 1个拷贝和2个拷贝菌株较多(21／74)。结论 目前“北京家族”结核分支杆菌菌株一定程度上在广东地区流行。     

ahpC基因突变与结核分支杆菌耐异烟肼的研究

目的了解我国结核分支杆菌耐异烟肼（ＩＮＨ）分离株ａｈｐＣ编码基因及其启动子突变情况，研究其与ＩＮＨ耐药关系。方法通过聚合酶链反应（ＰＣＲ）单链构象多态性（ＳＳＣＰ）方法分析６２株结核分支杆菌临床分离株ａｈｐＣ及其启动子基因。以Ｈ３７ＲＶ标准株为对照。结果３２株结核分支杆菌药物敏感株ａｈｐＣ及其启动子基因ＳＳＣＰ均泳动正常；３０株耐ＩＮＨ分离株ａｈｐＣ编码基因ＳＳＣＰ也未见异常；１２株高度耐ＩＮＨ分离株中，５株ａｈｐＣ启动子ＳＳＣＰ泳动异常；１８株低度耐ＩＮＨ分离株的ａｈｐＣ启动子泳动正常。结论结核分支杆菌ａｈｐＣ编码基因与耐ＩＮＨ无关；ａｈｐＣ启动子突变是结核分支杆菌ｋａｔＧ改变、过氧化氢酶缺乏的代偿性变化，也许能作为结核分支杆菌高度耐ＩＮＨ的间接指标。     

聚合酶链反应－单链构象多态性用于耐利福平结核分支杆菌rpoB基因突变的研究

目的研究结核分支杆菌ｒｐｏＢ基因突变及其与利福平（ＲＦＰ）耐药性的关系。方法以参考菌株结核分支杆菌Ｈ３７Ｒｖ为对照，用聚合酶链反应－单链构象多态性（ＰＣＲ－ＳＳＣＰ）方法分析了４０株结核分支杆菌临床分离株。ＰＣＲ－ＳＳＣＰ方法由ＰＣＲ扩增和扩增产物ＤＮＡ单链多态性分析组成。结果两对引物ＰＣＲ扩增产物分别为４１１和２５８ｂｐ，其敏感性分别为５ｐｇ／μｌ、５００个菌／ｍｌ和１ｐｇ／μｌ、５００个菌／ｍｌ；均为属特异性。４０株结核分支杆菌临床分离株２５８ｂｐ扩增片段ＳＳＣＰ图谱的特点：以参考菌株结核分支杆菌Ｈ３７Ｒｖ为对照，１０株敏感株均无区别；单耐ＲＦＰ或包括ＲＦＰ多种抗结核药３０株，除３株外，其余２７株ＳＳＣＰ图谱有明显的区别；检测阳性率为９０％，特异性为１００％。结论ＰＣＲ－ＳＳＣＰ方法可检测出耐ＲＦＰ结核分支杆菌ｒｐｏＢ基因突变；该基因是ＲＦＰ的药物靶编码基因，它的突变与ＲＦＰ耐药性有密切关系，这有助于结核分支杆菌耐药性的快速检测和研究     

DNA指纹技术方法学的建立及在结核分支杆菌菌株鉴定中的应用

目的探讨结核分支杆菌ＤＮＡ指纹技术的方法学及其在结核分支杆菌菌株鉴定中的应用。方法以结核分支杆菌染色体ＤＮＡ插入序列ＩＳ６１１０为基础,根据不同来源的结核分支杆菌菌株ＤＮＡ相异的ＩＳ６１１０拷贝数和相对分子质量,应用增强化学发光标记技术对结核分支杆菌ＤＮＡ酶切产物进行杂交和检测。结果不同来源的５０例结核患者临床分离株具有相异的ＤＮＡ指纹图谱。同一患者痰和膝关节脓培养阳性的结核分支杆菌菌株ＤＮＡ指纹图谱有较大的差异。人工诱导的结核分支杆菌Ｈ３７Ｒｖ氧氟沙星耐药株和敏感株有一致的ＤＮＡ指纹图谱。结论应用ＤＮＡ指纹技术进行结核分支杆菌株水平的鉴定是完全可行的     

吉林市结核分支杆菌IS6110DNA指纹图谱特征分析

目的：分析吉林市结核分支杆菌IS6110RFLP图谱特征。方法：对53株结核分支杆菌分离株进行IS6110基因分型，得到IS6110RFLP图谱；按菌株指纹特征的同源性高低予以分组，并进行分析。结果：吉林市结核分支杆菌IS6110RFLP图谱特征为：（1）IS6110拷贝数目平均为13个；（2）A、B两组同源性很高，具有“北京基因型”特征，C组多态性较强；（3）IS6110RFLP图谱特征分布无地域差异；（4）耐药菌株与敏感菌株图谱特征无明显差异，但初治耐药菌株成簇率较高。结论：吉林市结核分支杆菌IS6110RFLP图谱特征以“北京基因型”为主；但还具一些独有的特征。     

应用BACTEC 460 TB系统区分出的非结核分支杆菌进一步菌种鉴定意义的探讨

本文对 １９９１～１９９４年从临床痰标本中以 ＢＡＣＴＥＣ　ＮＡＰ法区分出并保留下来的５２株非结核分支杆菌进行了菌种鉴定，结果；鸟－胞内分支杆菌２５株（４８．１％）；龟－偶然分支杆菌６侏（１１．５％）；瘰疬分支杆菌４株（７．７％）；戈登分支杆菌８株（１５．４％）；微黄分支杆菌３株（５．８％）；结核分支杆菌４株（７．７％）；未定菌种２株（ ３．８％）。与此同时，对其中病历资料较为完整的％株菌株所对应的２９份病历与临床进行了核实。结果：确诊为肺结核病的１７例（株）；诊断为非结核分支杆菌病的７例（１４株）；另有５例（株）诊断为肺炎、癌症及稳定结核病灶等其它肺部疾病。由此可见，只有将菌种鉴定结果与相应的临床病例相结合，才具有实际意义。     

应用BACTEC 460 TB系统区分出的非结核分支杆菌进一步菌种鉴定意义的探讨

本文对１９９１￣１９９４年从临床痰标本中以ＢＡＣＴＥＣ ＮＡＰ法区分出并保留下来的５２株非结核分支杆菌进行了菌种鉴定，结果：鸟－胞内分支杆菌２５株（４８．１％）；龟－偶然分支杆菌６株（１１．５％）；瘰疬分支杆菌４株（７．７％）；戈登分支杆菌８株（１５．４％）；微黄分支杆菌３株（５．８％）；结核分支杆菌４株（７．７％）；未定菌种２株（３．８％）。与此同时，对其中病历资料较为完善的３６株菌株所对应的２     

噬菌体生物扩增法快速检测结核分枝杆菌方法学研究

目的建立噬菌体生物扩增法快速检测结核分枝杆菌试验方法,探讨最佳检测条件.方法应用分枝杆菌噬菌体感染结核分枝杆菌,比较各种检测条件对测定结果的影响,并将所建方法用于痰标本检测,结果与BIOTEC Lab公司产品比较.结果 1×109噬菌斑形成单位(PFU)/ml的噬菌体,37℃感染 60 min可检出200～500条/ml结核分枝杆菌.杀毒剂100 mmol /ml,室温作用5 min可完全灭活受试噬菌体.指示细胞浓度在1×108条/ml 时,形成的噬菌斑清晰,便于计数.加热灭活的细菌和指示细胞不被噬菌体感染.H37Rv、H37Ra和牛分枝杆菌参考菌株检测结果均为阳性,7种非分枝杆菌和16种常见非结核分枝杆菌检测结果为阴性,4种非结核分枝杆菌(偶然、胞内、金色、草分枝杆菌)在高浓度时(>1×105条/ml)检测结果为阳性.重复试验结果表明,本法批间和批内变异系数均<15%.痰标本氢氧化钠-半胱氨酸液化方式的阳性检出率为94%(49/52),显著高于氢氧化钠液化结果的62%(32/52,χ2＝15.06,P<0.01);预培养1 d的阳性检出率为65%(51/78),显著高于未预培养结果的40%(31/78,χ2＝18.05,P<0.01).临床标本检测结果表明,我们的试剂与进口试剂检测结果基本相同.结论噬菌体生物扩增法快速、简便、灵敏,有助于结核分枝杆菌的快速检测,但是测定条件的选择极为重要.     

Identification of Mycobacteria and measurement of whole cell fatty acids using a gas chromatography computer system

The technique of combination gas chromatography with computer used in measuring fatty acids of 28 species standard Mycobacteria was introduced in this study. The data of components of fatty acids in this genus and GC graphs with satisfaction were gained. Several peaks with good reproducibility were distinguished automatically, based on which, M. tuberculosis, M. bovis and other comment atypical Mycobacteria may be separated respectively. It was showed that this method have the characteristics of accurate, sensitive and rapid by means of the identification of clinical strains, also have certain practical value in clinical laboratory.     

Pyrazinamidase activity of Mycobacterium tuberculosis--a test of sensitivity to pyrazinamide

Pyrazinamidase activity has been found to correlate with pyrazinamide sensitivity in strains of Mycobacterium tuberculosis. In vitro sensitivity to pyrazinamide in acidified L?wenstein-Jensen medium, and pyrazinamidase activity by the Wayne method, were determined in 378 clinical isolates of M. tuberculosis. A close correlation was observed between the results of both tests. This method of detecting pyrazinamidase activity was found to be a rapid, simple and reliable substitute for pyrazinamide sensitivity testing, and it overcomes the difficulty of growing M. tuberculosis at pH 5.5, as required in the standard method.     

Evaluation of cycloserine in the treatment of infections caused by nontuberculous mycobacteria viewed from in vitro experiments

Evaluation of cycloserine as a drug in the treatment of infections caused by nontuberculous mycobacteria was made from in-vitro studies, in which Mycobacterium tuberculosis strains were used as the standard of the evaluation. The susceptibility testing to cycloserine was made using Ogawa egg medium. Bacterial suspensions, 10 mg wet weight/ml, prepared from 10 day-old cultures (M. tuberculosis, 14 day-old cultures) growing on Ogawa egg medium were used as the source of inoculation. A 0.02 ml-sample of the suspensions was inoculated onto Ogawa egg medium containing cycloserine or containing no drug, and the media inoculated were incubated at 37 degrees C. The minimal inhibitory concentration (MIC) was determined after incubation for 14 days (M. tuberculosis, for 21 days). The MIC was determined as the lowest concentration of the drug, on which the growth of test strains was completely inhibited. However, residual growth was sometimes observed. This was regarded as growth inhibition, because control medium containing no drug showed always abundant, membraneous growth. The results are shown in Fig.. The growth of M. tuberculosis strains was inhibited by the concentrations of 6.25 to 25 micrograms/ml. However, considering our previous observations on the relationship between the cycloserine resistance and the drug efficacy (reference 1), we regarded the MIC 12.5 micrograms/ml as critical concentration for presumable clinical efficacy. The ratios of strains of various mycobacterial species showing the MICs lower than the critical concentration are shown in Table. As seen in this table, clinical efficacy of cycloserine was expected in the treatment of infections caused by Mycobacterium kansasii, M. malmoense, M. simiae, M. scrofulaceum and M. marinum.(ABSTRACT TRUNCATED AT 250 WORDS)     

RNA恒温扩增实时检测技术快速鉴定偶发分枝杆菌的研究

目的建立RNA恒温扩增实时检测技术快速鉴定偶发分枝杆菌(RIARD-MF)的方法,评估其在鉴定分枝杆菌临床分离株中的应用效果。方法将偶发分枝杆菌的16SrRNA特异序列作为目的靶标进行检测,设计含T7启动子逆转录扩增引物和RNA探针,分别对5种非分枝杆菌细菌、20种分枝杆菌标准株和259株临床分枝杆菌分离株进行42℃恒温扩增实时检测,参考标准为PCR基因测序结果。结果RIARD-MF方法学检测灵敏度可达60CFU/mL。在25种细菌的RIARD-MF特异性检测中:只有偶发分枝杆菌检测结果为阳性,其余24种细菌均为阴性,与测序检测结果一致。在检测分枝杆菌临床分离菌株时,RIARD-MF鉴定偶发分枝杆菌5株,其余为非偶发分枝杆菌,与测序结果一致,检测灵敏度和特异度均为100%。结论 RIARD-MF鉴定偶发分枝杆菌具有较高的特异度、灵敏度和准确性,且检测快速,有望成为一种新的偶发分枝杆菌临床分离菌株鉴定方法。     

Microplate hybridization methods of reduced unspecific reassociation considering delta Tm--comparison between relative relatedness of DNA and key characters for Mycobacterium szulgai

It has been recognized that colorimetric microdilution plate hybridization method (DDH) shows equivocal identification results for some strains of Mycobacterium gordonae, and that chemotaxonomic identification method reveals some intermediate pattern between Mycobacterium szulgai and M. gordonae. In the present study, the results obtained by chemotaxonomic identification method on 25 strains of M. szulgai were compared with those obtained by DDH method. In chemotaxonomic methods, 8 of 25 M. szulgai strains showed negative results on 14 days' tween80 hydrolysis, 14 strains revealed negative nitrate reduction by the Tsukamura's method but all were positive by the Virtanen's method. Smooth colonies were found in 3 strains including M. szulgai type strain, JATA3201 (ATCC35799). Relative relatedness (relative color index) of genomic DNA was measured by DDH instead of spectrophotometric genomic DNA-DNA relatedness. A relative relatedness of 25 M. szulgai strains tested showed higher levels than 80%, but the inter-species relatedness to the other mycobacteria also showed high levels of 50-75%, when hybridizing temperature was set at 40 degrees C. At 56 degrees C, intra-species relative relatedness in 4 strains were lower than 50%, indicating that this condition is not appropriate. When hybridization temperature raised to 56 degrees C after overnight at 40 degrees C, a relative relatedness among 25 strains were again high (> 80%), and those to the other Mycobacterial species were lower than 70%. When hybridized at 56 degrees C after overnight at 45 degrees C, an intra-species relative relatedness again showed higher levels than 70% in all 25 strains, and interspecies percentiles were lowered satisfactorily to < 25%. In conclusion, through avoiding reassociation of nonspecific DNA fragments during the hybridization process, 45 degrees C overnight followed by 56 degrees C hybridization (delta DDH method) was found to be the better condition for identification and classification of Mycobacterium szulgai.     

Evaluation of the GonoGen II kit for rapid identification of Neisseria gonorrhoeae using monoclonal antibody directed at gonococcal outer membrane protein 1

The Gonogen II test for rapid identification of Neisseria gonorrhoeae (Gonococcus, GC) was evaluated. The test is based on a colorimetric reaction with monoclonal antibody to GC outer membrane protein 1. Of the 50 clinical isolates of GC, 49 isolates tested positive and only one strain tested negative. Other Neisseria. spp, H. influenzae, H. parainfluenzae, E. corrodens, M. catarrhalis, and A. baumannii showed negative test results. Non-Neisseriae. spp, such as S. aureus. P. aeruginosa, E. faecalis, and E. coli also showed negative test results. No cross-reactivity was found between GC and other Neisseriae. spp or non-Neisseriae. spp. In a mixed suspension of GC and all of non-Neisseriae. spp as mentioned above, the GonoGen II test was positive. The specificity and sensitivity of the test for the identification of GC were 98% and 100%. The minimum limit of detection of GC was > or = 1 x 10(5) cfu/mL. Decision making based on the test result is possible within 10 minutes. These findings also suggest that the test does not require pure GC. The GonoGen II test appears to be a reliable, quick and easy-to-use assay, and also to not require viable GC. Thus GonoGen II is shown to be a very useful test for the identification of GC.     

Profiling of genes expressed in peripheral blood mononuclear cells predicts glucocorticoid sensitivity in asthma patients

Gene expression profiles were examined in freshly isolated peripheral blood mononuclear cells (PBMC) from two independent cohorts (training and test sets) of glucocorticoid (GC)-sensitive (n = 64) and GC-resistant (n = 42) asthma patients in search of genes that accurately predict responders and nonresponders to inhaled corticosteroids. A total of 11,812 genes were examined with high-density oligonucleotide microarrays in both resting PBMC (106 patients) and cells treated in vitro with IL-1beta and TNF-alpha combined (88 patients), with or without GC. A total of 5,011 genes were expressed at significant levels in the PBMC, and 1,334 of those were notably up-regulated or down-regulated by IL-1beta/TNF-alpha treatment. The expression changes of 923 genes were significantly reversed in GC responders in the presence of GC. The expression pattern of 15 of these 923 genes that most accurately separated GC responders (n = 26) from the nonresponders (n = 18) in the training set, based on the weighted voting algorithm, predicted the independent test set of equal size with 84% accuracy. The expression accuracy of these genes was confirmed by real-time-quantitative PCR, wherein 11 of the 15 genes predicted GC sensitivity at baseline with 84% accuracy, with one gene predicting at 81% in an independent cohort of 79 patients. We conclude that we have uncovered gene expression profiles in PBMC that predict clinical response to inhaled GC therapy with meaningful accuracy. Upon validation in an independent study, these results support the development of a diagnostic test to guide GC therapy in asthma patients.     

一种直接检测结核病痰标本的PCR技术的建立与评价

目的建立并评价聚合酶链式反应(polymerase chain reaction,PCR)在结核病痰标本检测中的应用价值。方法根据结核分枝杆菌复合体IS6110序列设计引物INS1和INS2,并建立PCR反应体系和反应条件。运用PCR方法分别检测标准菌株、结核分枝杆菌PCR检测标准品和拟诊结核病患者痰标本,采用痰涂片和细菌培养为对照。结果比较的统计学分析采用卡方检验。结果PCR方法对结核分枝杆菌、牛分枝杆菌、卡介苗标准株的最小检出浓度分别达到102,103,103个细菌/毫升,能够特异地检出结核分枝杆菌复合体。在PCR检测的574例拟诊病例中,PCR检测阳性病例241例,42%;痰涂片和细菌培养的阳性率分别为19.69%和26.31%,PCR检测阳性率高于传统细菌学检验方法,经χ2检验,差异具有显著统计学意义(χ2=103.67,P<0.01)。以痰培养结果为标准,计算PCR检测方法的敏感度为67.53%。结论PCR检测方法与传统的细菌学检测方法相比可以提高阳性标本检出率,而且具有快速、特异、简便的特点,有望成为结核病大规模筛查和临床快速检测方法。     

牛分枝杆菌的噬菌体快速检测方法研究

建立牛分枝杆菌噬菌体快速检测技术,探讨最佳的检测条件,并将建立的方法用于牛分枝杆菌及16种常见非结核分枝杆菌和6种其他细菌的检测。结果表明,噬菌体工作浓度为1×108PFU/ml、37℃感染2h为最佳检测条件。杀毒剂浓度在100mmol/L,室温作用10min即可完全杀灭受试噬菌体。选择浓度为4mg/ml的对数生长期指示细菌为工作浓度,获得理想的检测结果。加热灭活的细菌和指示细菌不被噬菌体感染。牛分枝杆菌和耻垢分枝杆菌检测结果为阳性,16种常见非结核分枝杆菌和6种其他细菌检测结果均为阴性。该法可检测出60~120条/ml牛分枝杆菌。重复性试验表明,批内、批间变异系数均小于15%,重复性良好。