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1.
目的 评价IB6110-限制性片段长度多态性(IS6110-RFLP)和间隔区寡核苷酸分型技术(spoligotyping)两种方法在结核病流行病学上的应用,探讨我国各地区的结核分枝杆菌菌株特点。方法 收集158株结核分枝杆菌临床分离株,分别应用IS6110-RFLP和Spoligotyping两种方法进行鉴定。结果 (1)IS6110-RFLP的分辨力大于spoligotyping分型。(2)将本次试验结果与国际spoligotype数据库进行比较。结果 有14个类型属于共有类型,其中类型1为流行的类型,分布广泛,即所说的北京基因型。(3)广东地区与其他地区成簇率和北京基因型所占比例差异有统计学意义(P〈0.05)。广东地区成簇率和北京基因型所占比例均显著低于其他地区。结论 同时应用IS6110-RFLP分型和Spoligotyping两种方法进行结核病流行病学调查研究非常有效,在中国不同地区的菌株具有不同的特点。  相似文献   

2.
目的 评价多位点MLVA的15位点组合在北京基因型结核分枝杆菌(MTB)基因分型研究中的应用价值.方法 采用MLVA(15位点)对来源于北京胸科医院的72株北京基因型MTB菌株进行基因分型,并将结果与金标准IS6110-RFLP的分型结果进行比较.结果 MLVA(15位点)分型后共得到59个类型,其中53个为独特类型,其总体分辨力为0.990,多态性较好的位点有Qub-11b、Mtub 21和QUB-26;IS6110-RFLP分型后共得到69个类型,其中66个为独特类型,分辨力高达0.999,并可对MLVA(15位点)中成簇的菌株进一步区分.结论 MLVA(15位点)在北京基因型菌株中具有较好的分辨能力,但成簇菌株仍需采用金标准IS6110-RFLP进一步细分.  相似文献   

3.
结核分枝杆菌随机扩增多态性DNA指纹分型技术及应用研究   总被引:8,自引:1,他引:7  
目的 探讨重庆地区结核病分子流行病学的发展趋势。方法 建立随机扩增多态性DNA聚丙酰胺凝胶电泳(RAPD-PAGE)指纹分型法;采用1对随机引物对重庆地区287例肺结核患者416株结核分枝杆菌进行DNA指纹分型。结果 416株分离菌株其DNA指纹可分为7种类型,以Ⅰ型(35.5%)、Ⅱ型(29.3%)和Ⅲ型(22.6%)为主,其中20-39岁和40-49岁年龄组的初治患者在3型中分别占58.0%和46.2%.在Ⅰ、Ⅱ、Ⅲ型中初治患者分别占52.0%、46.4%和58.5%,至少耐利福平和异烟肼(MDR)的初治患者又分别占的11.3%、35.9%和24.3%,至少耐利福平和其他药物的多理耐药株在3型中占21.7%(初治)。结论 RAPD-PAGE指纹分型法,是一种简便、特异、敏感、重复性好的菌株分型方法,可用于结核病分子流行学的研究;以Ⅰ、Ⅱ、Ⅲ型为主的结核分枝杆菌正在重庆及周边地区的人群中传播;MDR株和含利福平的其他多种耐药株的传播占有较高频率。  相似文献   

4.
目的初步了解中国部分地区结核分枝杆菌临床分离菌株数目可变串联重复序列(variable number of tandem repeats,VNTRs)基因多态性特征。 方法采用多位点VNTRs分析(multiple loci VNTRS analysis, MLVA)技术,随机选取159株中国部分地区结核分枝杆菌临床分离菌株,PCR和琼脂糖凝胶电泳技术,对结核分枝杆菌的19个VNTRs位点进行检测,用BioNumerics (Version 5.0) 软件进行结果分析,数据结果与国际MLVA数据库(http://minisatellites.u-psud.fr)比对,初步分析结核分枝杆菌DNA 多态性特征。结果159株菌株大致被分成4个主要的簇,其中73.8%的菌株为北京家族菌株,其次为H37Rv相似菌株及在数据库中没有比对结果的一簇,这一簇的MLVA特点是ETRD3,MIRU10、MIRU27、MIRU39、MIRU40及Mtub21位点结果为22221,与其他簇的菌株结果有比较明显的区别。可能是中国特有的菌株。另外发现与BCG相似的一簇。结论本次研究中的中国结核分枝杆菌具有明显的VNTR基因多态性,主要流行菌株为北京家族菌株,可能存在中国特异的VNTR基因型。BCG临床分离株的发现,提示BCG疫苗相关病例可能成为值得关注的卫生问题。  相似文献   

5.
目的 用间隔区寡核苷酸分型(Spoligotyping)鉴定杭州结核分枝杆菌临床分离株的基因分型种类和特征,为结核病防治研究提供基础科学依据。方法 收集结核分枝杆菌杭州临床分离株,应用Spoligotyping进行基因分型检测。基因聚类分析采用BioNumerics(Version 5.0)软件。结果 共在杭州市收集到99株结核分枝杆菌临床分离株,可分为2个基因群,即北京家族(Beijing family)和非北京家族(Non?鄄Beijing family),分别占64.65%(64/99)和35.35%(35/99),20种基因型,其中9株为独特类型,剩余90株分为11簇。北京家族菌株中,89.06%(57/64)为典型北京家族(Typical Beijing family),35株非北京家族菌株可分为16个基因型,10株为独特的基因型。结论 杭州结核分枝杆菌存在明显的基因多态性,北京家族菌株占优势,但非北京家族菌株基因多态性更显著。  相似文献   

6.
摘要:目的:建立基于gyrB基因扩增检测结核分枝杆菌复合物(MTC)的荧光定量PCR(FQ-PCR)法。方法:根据gyrB基因设计合成引物和探针,建立并评价TaqMan探针FQ-PCR法,并与基于IS6110序列的FQ-PCR法分别检测50例临床标本,比较两种方法的检测结果。结果:建立的FQ-PCR法标准曲线方程为Y=-3.18X+42.84,相关系数(r2)为0.995、扩增效率为106.25%,其检测灵敏度为102) CFU/mL;MTC中结核分枝杆菌和非洲分枝杆菌阳性,其他分枝杆菌和4株临床其他细菌均为阴性。批内及批间变异系数(CV)均<2%,重复性良好。本法与基于IS6110序列的FQ-PCR法对50例临床标本的检测结果差异无统计学意义(χ2)=0.06,P>0.05),一致性好(κ=0.94)。结论:基于gyrB基因扩增的FQ-PCR法灵敏度高、特异性强、重复性好,可用于结核分枝杆菌复合物的早期快速诊断。  相似文献   

7.
目的分析3起由副溶血弧菌(VP)导致的食源性疾病暴发事件,利用国家致病菌识别网(China PIN)对其进行关联性分析。方法收集2018年8月北京市顺义区3起食源性疾病暴发事件的流行病学资料,采集可疑食品、环境和病例样本48份,进行菌株分离和鉴定,对分离的VP菌株进行脉冲场凝胶电泳(PFGE)分子分型,获得的指纹图谱通过致病菌识别网进行比对分析。结果3起暴发事件中,共从12份病例样本和3份可疑食品样本中分离出15株O3:K6血清型VP,其种特异性基因和毒力基因检测结果为:toxR+/tdh+/trh-,根据PFGE指纹图谱可以将15株VP分成2个簇,每个簇中的指纹图谱相似度为100%。 2个指纹图谱簇之间的相似度为93.94%;且与2014 — 2017年本地区China PIN分子分型数据库中分离自腹泻病例的VP菌株带型不成簇。结论VP引起的3起食源性疾病暴发事件由共同的传染源导致,China PIN在食源性疾病暴发识别、溯源和关联性分析中有良好的应用前景。  相似文献   

8.
目的利用肠杆菌基因间重复序列为引物的聚合酶链反应(enterbacterial repetitive intergenic consensus-PCR,ERIC-PCR)分型技术分析我市结核分枝杆菌流行情况。方法收集2003年9月至2006年5月门诊痰菌阳性标本,培养并提取DNA做ERIC—PCR指纹图,并利用RAPDPHYLIP及Treeview软件对其进行聚类分析。结果122株临床分离株产生42种不同的指纹图,聚类分析结果显示可分为3簇,成簇率76、2%。指纹图谱与患者年龄、菌株的耐药性及耐药种类有关。结论ERIC—PCR具有不需已知核酸序列,分辨效果好,简便快捷等优点,是进行分子流行病学调查的有效工具。我市结核病呈高水平传播,主要为耐药菌株并以中青年患者传播为主。  相似文献   

9.
目的 用免疫色谱法抗-MPB64单克隆抗体快速检测和诊断结核分枝杆菌结核菌群的方法学评价。方法 共收集20株临床标本分离菌株、11株参考菌株和1株结核分枝杆菌标准菌株,应用免疫色谱法检测在培养基上生长的细菌,并和传统鉴定方法、实时荧光探针定量PCR法(FQ-PCR)作比较研究。结果 用免疫色谱法检测1株标准菌株为阳性,检测11株参考菌株发现用该法能完全区分结核和非结核分枝杆菌。对20株临床分离的标本用免疫色谱检出11株结核菌菌群,检出率为55%;用传统鉴定方法检出10株,其中未能检出的一株为混合菌感染;用FQ-PCR法检出10株,其中未能检出的一株为牛结核菌。免疫色谱法能检测到的最低菌浓度为10^5CFU/ml。免疫色谱法、FQ-PCR法和传统鉴定方法的平均耗时分别为15min,1~2d和30d。结论 免疫色谱法是一种简便、快速、准确、敏感和特异性鉴别结核和非结核分枝杆菌的方法,适合在临床推广使用。  相似文献   

10.
目的确定杂交信号放大方法(rlSAM)检测结核分枝杆菌的灵敏度和特异性,进而探讨该方法用于检测临床标本的可行性。方法HSAM法检测合成的IS6110靶基因、结核分枝杆菌标准菌株、大肠埃希菌和表皮葡萄球菌。确定该方法的灵敏度和特异性,并与PCR法进行比较。结果HSAM最少能检测到10合成的靶基因和结核分枝杆菌,与PCR方法灵敏度相近,标准菌株检测结果阳性,大肠埃希菌和表皮葡萄球菌均为阴性,该方法具有较高的特异性。结论HSAM具有高灵敏度、高特异性及操作简便快速等特点,可作为结核分枝杆菌临床标本检测方法。  相似文献   

11.
Isoniazid (INH) is a central component of drug regimens used worldwide to treat tuberculosis. Previous studies have identified resistance-associated mutations in katG, inhA, kasA, ndh, and the oxyR-ahpC intergenic region. DNA microarray-based experiments have shown that INH induces several genes in Mycobacterium tuberculosis that encode proteins physiologically relevant to the drug's mode of action. To gain further insight into the molecular genetic basis of INH resistance, 20 genes implicated in INH resistance were sequenced for INH resistance-associated mutations. Thirty-eight INH-monoresistant clinical isolates and 86 INH-susceptible isolates of M. tuberculosis were obtained from the Texas Department of Health and the Houston Tuberculosis Initiative. Epidemiologic independence was established for all isolates by IS6110 restriction fragment length polymorphism analysis. Susceptible isolates were matched with resistant isolates by molecular genetic group and IS6110 profiles. Spoligotyping was done with isolates with five or fewer IS6110 copies. A major genetic group was established on the basis of the polymorphisms in katG codon 463 and gyrA codon 95. MICs were determined by the E-test. Semiquantitative catalase assays were performed with isolates with mutations in the katG gene. When the 20 genes were sequenced, it was found that 17 (44.7%) INH-resistant isolates had a single-locus, resistance-associated mutation in the katG, mabA, or Rv1772 gene. Seventeen (44.7%) INH-resistant isolates had resistance-associated mutations in two or more genes, and 76% of all INH-resistant isolates had a mutation in the katG gene. Mutations were also identified in the fadE24, Rv1592c, Rv1772, Rv0340, and iniBAC genes, recently shown by DNA-based microarray experiments to be upregulated in response to INH. In general, the MICs were higher for isolates with mutations in katG and the isolates had reduced catalase activities. The results show that a variety of single nucleotide polymorphisms in multiple genes are found exclusively in INH-resistant clinical isolates. These genes either are involved in mycolic acid biosynthesis or are overexpressed as a response to the buildup or cellular toxicity of INH.  相似文献   

12.
In the present study, 77 drug-resistant Mycobacterium tuberculosis strains isolated in Poland in 2000 were characterized by the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and our novel method based on PCR amplification of DNA regions between IS6110 and 16-bp GC-rich frequent repeats (designated IS6110-Mtb1/Mtb2 PCR). The results were compared with previous data of the more commonly used methods, IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The discriminatory power of IS6110-Mtb1/Mtb2 method was only slightly lower than that of IS6110 RFLP, whereas MIRU-VNTR typing was the least discriminative among the 4 methods used. Clustering of strains by using results of IS6110-Mtb1/Mtb2 PCR correlated well with RFLP-defined clusters, further confirming epidemiologic relationships among patients. These results indicate that the novel genotyping method could be an attractive alternative for other PCR-based typing procedures, such as spoligotyping and MIRU-VNTR typing. Also, it seems to be a valuable adjunct to the reference IS6110 RFLP method for studying the genetic diversity of drug-resistant M. tuberculosis strains in Poland.  相似文献   

13.
目的 了解13株卡介苗(bacille calmette-gurin, BCG)基因组中由PE/PPE基因家族编码的B细胞抗原表位分布情况, 分析其对BCG免疫保护力的潜在影响。方法 从国际免疫表位数据库(Immune Epitope Data base, IEDB)中检索结核分枝杆菌B细胞抗原表位, 与结核分枝杆菌参考菌株H37Rv的蛋白质组进行序列比对, 确定PE/PPE基因家族编码B细胞抗原表位序列;并与13株BCG基因组进行序列比对, 提取表位编码序列, 分析其在BCG中的分布与变异。结果 6个PE/PPE基因家族的基因编码28个B细胞抗原表位, 21个B细胞抗原表位在13株BCG中高度保守, 7个B细胞抗原表位在BCG中发生基因变异, 变异表位分别产生于BCG的不同形成期。结论 PE/PPE基因家族所表达的蛋白大多为细菌表面蛋白, PE/PPE蛋白家族中B细胞抗原表位的变化可能对BCG的保护力产生影响, 应继续开展基于B细胞表位的BCG遗传特征研究。  相似文献   

14.
Multiple copies of an insertion sequence, IS6110, were shown to be present in the genome of members of the Mycobacterium tuberculosis complex (M. tuberculosis and M. bovis). Ten to 12 copies are present in various strains of M. tuberculosis, while strains of M. bovis contain only one to three copies. IS6110 was not detected in the DNA of other species of mycobacteria. Restriction endonuclease analysis indicated that the sequence of IS6110 is conserved across strain and species lines. Hybridization to the insertion sequence can be used to detect restriction fragment length polymorphism reflecting divergence in the sequence of regions flanking the various copies of IS6110. These differences were used to fingerprint various strains of the M. tuberculosis complex.  相似文献   

15.
OBJECTIVES: To characterize the efflux pump encoded by the gene Rv2333c from Mycobacterium tuberculosis, and assess its contribution to intrinsic antibiotic resistance using Mycobacterium bovis BCG as a model organism. METHODS: Firstly, the Rv2333c gene was expressed from a multicopy plasmid in M. bovis BCG. Secondly, the gene was inactivated in the chromosome of M. bovis BCG. Antibiotic susceptibility tests and tetracycline uptake/efflux experiments were carried out with the strains mentioned above. RESULTS: When the Rv2333c gene was inactivated in the M. bovis BCG chromosome, there was a decrease in the MIC values of spectinomycin and tetracycline, and an increase in [3H]tetracycline accumulation. When the Rv2333c gene was cloned into a multicopy plasmid, there was an increase in the MIC values of spectinomycin and tetracycline, and a decrease in [3H]tetracycline accumulation. These results indicate that both antibiotics are substrates of the Rv2333c efflux pump, which has been named Stp, for Spectinomycin Tetracycline efflux Pump. CONCLUSIONS: The Rv2333c efflux pump (Stp protein) of M. tuberculosis contributes to intrinsic spectinomycin and tetracycline resistance.  相似文献   

16.
Isoxyl (ISO), a thiourea (thiocarlide; 4, 4'-diisoamyloxythiocarbanilide), demonstrated potent activity against Mycobacterium tuberculosis H37Rv (MIC, 2.5 micrograms/ml), Mycobacterium bovis BCG (MIC, 0.5 microgram/ml), Mycobacterium avium (MIC, 2.0 microgram/ml), and Mycobacterium aurum A+ (MIC, 2.0 microgram/ml), resulting in complete inhibition of mycobacteria grown on solid media. Importantly, a panel of clinical isolates of M. tuberculosis from different geographical areas with various drug resistance patterns were all sensitive to ISO in the range of 1 to 10 microgram/ml. In a murine macrophage model, ISO exhibited bactericidal killing of viable intracellular M. tuberculosis in a dose-dependent manner (0.05 to 2.50 microgram/ml). The selective action of ISO on mycolic acid synthesis was studied through the use of [1, 2-14C]acetate labeling of M. tuberculosis H37Rv, M. bovis BCG, and M. aurum A+. At its MIC for M. tuberculosis, ISO inhibited the synthesis of both fatty acids and mycolic acids (alpha-mycolates by 91.6%, methoxymycolates by 94.3%, and ketomycolates by 91.1%); at its MIC in M. bovis BCG, ISO inhibited the synthesis of alpha-mycolates by 87.2% and that of ketomycolates by 88.5%; and the corresponding inhibitions for M. aurum A+ were 87.1% for alpha-mycolates, 87.2% for ketomycolates, and 86.5% for the wax-ester mycolates. A comparison with isoniazid (INH) and ethionamide (ETH) demonstrated marked similarity in action, i.e., inhibition of the synthesis of all kinds of mycolic acids. However, unlike INH and ETH, ISO also inhibited the synthesis of shorter-chain fatty acids. ISO showed no acute toxicity against primary macrophage cell cultures as demonstrated by diminution of redox activity. A homologous series of ISO derivatives were synthesized. Most derivatives were as effective or more effective than the parent compound in the agar proportion assay. Thus, these thioureas, like INH and ETH, specifically inhibit mycolic acid synthesis and show promise in counteracting a wide variety of drug-sensitive and -resistant strains of M. tuberculosis.  相似文献   

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