MLVA和Spoligotyping用于西藏地区216株结核分枝杆菌临床分离株的基因分型研究

目的评价间隔区寡核苷酸分型（Spoligotyping）及多位点可变数量串联重复序列分析（MLVA）两种分型方法在西藏地区结核病分子流行病学中的应用。方法收集西藏地区结核分枝杆菌（Mycobacteriumtuberculosis）临床分离株，应用Spoligotyping及MLVA两种分型方法进行比较分析。结果共在西藏地区收集到216株结核分枝杆菌临床分离株，采用Spoligotyping分型方法，216株菌可分为3个基因群13种基因型，其中最大的1个基因群即北京家族（8eijingfamily）含有195株菌，占90．28％。北京家族菌株中，有BCG接种史者占45．64％（89／195），无BCG接种史者占54．36％（106／1195），两者间的差异无统计学意义（X^2=0．059，P〉0．05）。采用MLVA分型方法，216株菌可分成19个基因群108种基因型，其中80种基因型只有1株菌，占37．03％（80／216），另有136株菌表现出28种基因型，成簇数为28，占62．96％（136／216）。在20个VNTR位点的等位基因多态性发现Miru31位点的多态性最高，多态性指数（h）达到0．77，而Mtub29、Mtubl2位点的多态性较差，都低于0．05。其中Mtuh02位点可鉴别北京家族和非北京家族，它鉴别的北京家族与Spoligotyping鉴别的北京家族符合率达到100％。结论西藏地区结核分枝杆菌具有明显的接引多态性，其主要流行型为北京家族。北京家族菌株与BCG接种无相关性。应用Spoligotyping和MLVA两种分型方法进行结核病流行病学研究，将提高结核病的流行病学调查和病原学监测效果。     

用PFGE鉴定结核分枝杆菌与非结核分枝杆菌

快速、准确地鉴定结核分枝杆菌与非结核分枝杆菌（MOTF）对结核病与非结核分枝杆菌病的诊断与治疗具有重要的指导意义。本研究以传统鉴定法为“金标准”，对94株分枝杆菌进行了分析，基于脉冲场凝胶电泳（pulsed-field gel electrophoresis．PFGE）及聚类分析的原理，建立了鉴定结核分枝杆菌与MOTF的新方法，并对其进行了方法学评价。     

分子信标用于结核分枝杆菌的均相荧光PCR检测

1996年Ｔｙａｇｉ等〔1〕提出的分子信标是一种具有颈环构型的分子探针 ,用于ＰＣＲ扩增产物均相测定的原理是 ,在退火阶段 ,分子信标与生成的靶序列结合发出荧光 ,在延伸阶段 ,则脱离靶序列而不干扰扩增 ,随着循环次数的增加 ,与模板结合的分子信标的量亦增加 ,最终的荧光强度便与模板量成正相关。我们在原来“分子信标”原理基础上 ,对其设计思想进行了重要改进 ,合成出效率更高的分子信标 ,并自行研制了简便适用的荧光测定装置。本文报告用于结核分枝杆菌的检测结果。材料和方法菌株 :分枝杆菌属包括堪萨斯分枝杆菌、瘰疬分枝杆菌、耻垢…     

结核分枝杆菌临床分离株异烟肼耐药相关基因突变的分子特征

目的 阐明结核分枝杆菌异烟肼（INH）耐药相关基因突变特征.方法 对137株结核分枝杆菌临床分离株（耐异烟肼菌株87株,异烟肼敏感菌株90株）的9个结构基因furA、katG、inhA、kasA、Rv0340、iniB、iniA、iniC和efpA以及两个调控区oxyR-ahpC基因间隔区和mabA-inhA启动子进行DNA片段扩增及序列分析.结果 82株（94.3%）INH耐药分离株的katG基因存在突变,其中katGSer315Thr突变占优势（55.2%）.50株INH敏感的分离菌katG的463密码子没有突变.35株（40.2%）INH耐药的分离株katG的463有突变.87株INH耐药株中,20株（23.0%）的katG基因存在两重突变.13株（14.9%）分离菌inhA基因的启动子区存在突变,4.6%的分离菌有inhA结构基因突变,11.5%oxyR-ahpC基因间区存在突变.iniBAC区域和efpA中发现耐药性关联突变.结论 研究证实多个基因突变与异烟肼耐药之间的关系,并且为阐明结核分枝杆菌耐药机制提供线索.     

新疆南疆地区维吾尔族结核病患者结核分枝杆菌临床分离株的基因分型研究

目的 应用多位点数目可变串联重复序列(VNTR)分析技术,对新疆南疆地区维吾尔族结核病患者结核分枝杆菌临床分离株进行基因分型,探讨其数目VNTR基因型种类及其分布.方法 收集结核分枝杆菌,采用PCR和琼脂糖凝胶电泳技术,结合BioNumerics 5.0软件,对其24个VNTR位点进行结果分析.结果 分离出151株结核分枝杆菌,分为8个基因群151个基因型,其中Ⅵ群为主要基因群,占44.4%,有67个基因型,其次是Ⅷ群(23.2%)和Ⅳ群(20.5%).结论 新疆南疆地区维吾尔族结核病患者的结核分枝杆菌存在明显基因多态性,且存在主要流行菌群.     

结核分枝杆菌抗原分析及免疫交叉反应研究

目的：筛选和鉴定结核分枝杆菌特异性和保护性抗原，研究结核杆菌的免疫反应特点，以探索结核病诊断和治疗的新途径。方法：采用超声破碎和滤膜抽滤的方法分别得到菌体蛋白和滤液蛋白，通过Western blot试验用结核杆菌的单克隆抗体及结核病人血清来检测蛋白样品，把发生阳性反应的蛋白在BECKMAN LF3200／多肽氨基酸序列测定仪上进行N末端序列分析，并用结核杆菌的单抗对自身抗原组蛋白进行了检测。结果：结核杆菌的31kD和30kD蛋白与结核杆菌的单抗及病人血清反应均呈阳性，但与正常小鼠血清和健康人血清反应呈阴性。31kD和30kD蛋白的N末端序列分别为：Ala Glu Val Asp Trp Leu Val Phe Ala Val和Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr。结核分枝杆菌的单抗与自身抗原组蛋白能发生免疫交叉反应。结论：结核杆菌的31kD和30kD蛋白是免疫保护性抗原，对免疫交叉反应分子基础的进一步研究必将增加对结核免疫机理的了解。     

VNTR技术用于安徽省耐药结核分支杆菌基因分型的初步研究

目的 探讨VNTR（variable number of tandem repeat）技术在安徽省耐药结核分支杆菌基因分型中的应用。方法初步选取13个分型效果较好的VNTR基因位点。应用聚合酶链反应（PCR）和琼脂糖凝胶电泳，建立检测耐药结核分支杆菌DNA指纹多态性的方法，分析耐药结核分支杆菌DNA多态性。结果共对78株耐药结核分支杆菌的13个VNTR位点进行了检测，根据这些菌株的指纹多态性特征，共分为4个基因型（Ⅰ型、Ⅱ型、Ⅲ型、Ⅳ型），分别为Ⅰ型3．8％（3／78）、Ⅱ型6．4％（5／78）、Ⅲ型10．3％（8／78）、Ⅳ型所占比例最大，为79．5％（62／78）。在Ⅳ型菌中，耐药菌株主要为耐多药（结核分支杆菌至少耐异烟肼和利福平两种药）和单耐利福平菌株，其所占比例分别为45．8％和22．6％。结论本资料分析表明，安徽省耐药结核分支杆菌的传播似以Ⅳ型菌株为主，应加强此型菌株流行的监控。     

采用基因打靶技术构建结核分枝杆菌新基因Rv0901缺失株的研究

目的：利用基因打靶技术构建基因打靶载体和基因缺失株，用于结核分枝杆菌基因Rv0901功能的研究。方法：结核分枝杆菌标准株H37Rv体外培养，对其Rv0901基因及两侧序列进行体外扩增，连接载体及目的片段，切除目的基因，再引入筛选标志构建重组自杀质粒，分别用酶切及PCR鉴定；用电穿孔法将重组自杀质粒转入结核杆菌H37Rv株，用PCR鉴定。结果：经鉴定PCR产物及插入片段大小与预期值相符，且为所需目的基因片段，成功切除靶片段；蓝白斑筛选证实标记基因插入片段插入方向正确；Rv0901基因缺失株用PCR鉴定成功缺失了目的基因片段2.5 kb。结论：成功构建了用于结核分枝杆菌基因打靶的置换型载体和Rv0901新基因缺失株，为Rv0901基因功能的研究奠定了基础。     

结核分枝杆菌的ESX致病系统和EspR蛋白

近几年,结核分枝杆菌卷土重来,再次成为威胁人类健康的致病菌之一。尽管很多ESX系统的致病因子和毒力因子被研究发现,但迄今为止,结核分枝杆菌致病机制仍然不是很清楚。EspR(Rv3849)蛋白的研究可能为结核病的预防及治疗提供新的策略。     

深圳地区结核分枝杆菌耐药分离株分子特征与表型特征相关性研究

目的 研究深圳地区2007-2008年分离的结核分枝杆菌耐药株分子特征与表型特征的相关性.方法 参照WHO/IUATLD标准,使用L-J药敏培养基,1%比例法药敏试验筛选针对异烟肼、利福平、链霉素、氧氟沙星、卡那霉素5种药物耐药或敏感的临床分离株,通过PCR扩增经筛选菌株的rpoB、katG、rpsL、rrs1、gyrAB、rrs2基因的相关序列,运用DNAStar和BLASTN进行序列分析,应用二倍稀释法测定其表型最低抑菌浓度(MIC)值.结果 筛得实验菌株123株,其中耐药株73株,全敏感株50株.异烟肼耐药株katG基因突变率为84.6%,突变位点全部为S315T或S315N.利福平耐药株rpoB基因突变率为93.6%,突变位点主要集中在S531L(30/44,68.2%)和H526D(9/44,20.5%)或H526R(1/44,2.3%).链霉素耐药株以rpsl基因突变为主,突变位点为K43R(19/27,70.4%)和K88Q(6/27,22.2%);rrs1基因突变较少见,仅491C→T(2/27,7.4%)及513A→C(1/27,3.7%);2个基因突变率合计为65.9%.氧氟沙星耐药株突变率为100%,以gyrA基因突变为主,突变位点包括D94A(2/11,18.2%)、S91P(4/11,36.4%)和A90V(3/11,27.3%),3个突变位点总突变率为81.8%(9/11);S95T存在于所有氧氟沙星耐药株及部分全敏感株gyrA基因中;gyrB未发现突变.卡那霉索耐药株rrs2基因突变率为61.1%;突变位点为1400 A→C(9/11,81.8%)和1483 G→T(2/11,18.2%).其他突变位点在全敏感株中未发现.临床耐药株同一耐药组别所包含耐药类型不同,相关耐药基因的突变位点相同,其MIC值基本一致;相关耐药基因突变位点不同,其MIC值存在明显差异.结论 结核分枝杆菌耐药基因突变存在一定的地域特性,表型特征因耐药基因突变位点不同存在耐药程度差异.     

The discovery,function and development of the variable number tandem repeats in different Mycobacterium species

Abstract

The method of genotyping by variable number tandem repeats (VNTRs) facilitates the epidemiological studies of different Mycobacterium species worldwide. Until now, the VNTR method is not fully understood, for example, its discovery, function and classification. The inconsistent nomenclature and terminology of VNTR is especially confusing. In this review, we first describe in detail the VNTRs in Mycobacterium tuberculosis (M. tuberculosis), as this pathogen resulted in more deaths than any other microbial pathogen as well as for which extensive studies of VNTRs were carried out, and then we outline the recent progress of the VNTR-related epidemiological research in several other Mycobacterium species, such as M. abscessus, M. africanum, M. avium, M. bovis, M. canettii, M. caprae, M. intracellulare, M. leprae, M. marinum, M. microti, M. pinnipedii and M. ulcerans from different countries and regions. This article is aimed mainly at the practical notes of VNTR to help the scientists in better understanding and performing this method.     

Molecular typing of Mycobacterium tuberculosis based on variable number of tandem DNA repeats used alone and in association with spoligotyping

Fingerprinting based on variable numbers of tandem DNA repeats (VNTR), a recently described methodology, was evaluated for molecular typing of Mycobacterium tuberculosis in an insular setting. In this study, VNTR fingerprinting was used alone or as a second-line test in association with spoligotyping, double-repetitive-element PCR (DRE-PCR), and IS6110 restriction fragment length polymorphism (RFLP) analysis, and the discriminatory power for each method or the combination of methods was compared by calculating the Hunter-Gaston discriminative index (HGI). The results obtained showed that in 6 out of 12 (50%) cases, VNTR-defined clusters were further subdivided by spoligotyping, compared to 7 out of 18 (39%) cases where spoligotyping-defined clusters were further subdivided by VNTR. When used alone, VNTR was the least discriminatory method (HGI = 0.863). Although VNTR was significantly more discriminatory when used in association with spoligotyping (HGI = 0.982), the combination of spoligotyping and DRE-PCR (HGI = 0.992) was still the most efficient among rapid, PCR-based methodologies, giving results comparable to IS6110 RFLP analysis. Nonetheless, VNTR typing may provide additional phylogenetical information that may be helpful to trace the molecular evolution of tubercle bacilli.     

Genetic diversity of Mycobacterium tuberculosis in Sicily based on spoligotyping and variable number of tandem DNA repeats and comparison with a spoligotyping database for population-based analysis

In a previous study, we proposed to associate spoligotyping and typing with the variable number of tandem DNA repeats (VNTR) as an alternative strategy to IS6110-restriction fragment length polymorphism (RFLP) for molecular epidemiological studies on tuberculosis. The aim of the present study was to further evaluate this PCR-based typing strategy and to describe the population structure of Mycobacterium tuberculosis in another insular setting, Sicily. A collection of 106 DNA samples from M. tuberculosis patient isolates was characterized by spoligotyping and VNTR typing. All isolates were independently genotyped by the standard IS6110-RFLP method, and clustering results between the three methods were compared. The totals for the clustered isolates were, respectively, 15, 60, and 82% by IS6110-RFLP, spoligotyping, and VNTR typing. The most frequent spoligotype included type 42 that missed spacers 21 to 24 and spacers 33 to 36 and derived types 33, 213, and 273 that, together represented as much as 26% of all isolates, whereas the Haarlem clade of strains (types 47 and 50, VNTR allele 32333) accounted for 9% of the total strains. The combination of spoligotyping and VNTR typing results reduced the number of clusters to 43% but remained superior to the level of IS6110-RFLP clustering (ca. 15%). All but one IS6110-defined cluster were identified by the combination of spoligotyping and VNTR clustering results, whereas 9 of 15 spoligotyping-defined clusters could be further subdivided by IS6110-RFLP. Reinterpretation of previous IS6110-RFLP results in the light of spoligotyping-VNTR typing results allowed us to detect an additional cluster that was previously missed. Although less discriminative than IS6110-RFLP, our results suggest that the use of the combination of spoligotyping and VNTR typing is a good screening strategy for detecting epidemiological links for the study of tuberculosis epidemiology at the molecular level.     

Pattern and diversity of cytokine production differentiates between Mycobacterium tuberculosis infection and disease

Tuberculosis (TB) remains a global health problem. The solution involves development of an effective vaccine, but has been limited by incomplete understanding of what constitutes protective immunity during natural infection with Mycobacterium tuberculosis. In this study, M. tuberculosis‐specific responses following an overnight whole‐blood assay were assessed by intracellular cytokine staining and luminex, and compared between TB cases and exposed household contacts. TB cases had significantly higher levels of IFN‐γ⁺TNF‐α⁺IL‐2⁺CD4⁺T cells compared with contacts. TB cases also had a significantly higher proportion of cells single‐positive for TNF‐α, but lower proportion of cells producing IL‐2 alone and these differences were seen for both CD4⁺and CD8⁺ T cells. Cytokine profiles from culture supernatants were significantly biased toward a Th1 phenotype (IFN‐γ and IL‐12(p40)) together with a complete abrogation of IL‐17 secretion in TB cases. Our data indicate that despite a robust response to TB antigens in active TB disease, changes in the pattern of cytokine production between TB infection and disease clearly contribute to disease progression.     

Spoligotype patterns of Mycobacterium tuberculosis isolated from extra pulmonary tuberculosis patients in Puducherry,India

Purpose: Genotyping studies like spoligotyping are valuable tools in understanding the genetic diversity and epidemiology of Mycobacterium tuberculosis. Though there are reports of spoligotyping of M. tuberculosis isolates from pulmonary specimens from different parts of India, spoligotyping of extra pulmonary tuberculosis isolates are very few. Puducherry has not yet recorded spoligopatterns of M. tuberculosis from either pulmonary or extra pulmonary (EPTB) specimens. The aim of this study is to analyze the spoligotype patterns of EPTB strains circulating in Puducherry and neighboring districts of Tamil Nadu. Materials and Methods: During June 2011 to December 2013, 570 EPTB specimens were processed by culturing on to Lowenstein Jensen (LJ) medium and automated Mycobacterium Growth Indicator Tube system (MGIT960). Identification of M. tuberculosis was carried out as per standard procedures, and MPT 64 antigen positivity in a commercial immunochromatography kit. Spoligotyping was carried out at National Institute of Research in Tuberculosis (ICMR), Chennai. Results: M. tuberculosis was isolated from 67 single EPTB specimens (11.8%) like pus/cold abscess (34), TB spine (10), pleural fluid (10), urine (5), tissue bit (2), lymph nodes (2), ascitic fluid (2), synovial fluid (1) and endometrial curetting (1). Among 67 isolates with 41 spoligopatterns, EAI lineage with 28 isolates (41.8%) predominated followed by 18 orphans (26.9%), 10 Beijing (14.9%) and 8 U (11.9%). BOVIS1_BCG (ST482), T1-T2 (ST78) and H3 (ST50) were represented by one strain each (1.5%). Conclusions: Spoligotyping plays a significant role in the epidemiology of tuberculosis. Three spoligotypes, T1-T2 (ST78), EAI6 (ST292) and U (ST1429) are reported for the first time in India.     

Epidemiological studies of Beijing strains of Mycobacterium tuberculosis from Taipei and other Asian cities based on MIRU profiles

The aim of the present study was to investigate the distribution of the Beijing strains of Mycobacterium tuberculosis (MTB) in Taipei and other Asian cities. A total of 323 MTB isolates were analyzed by spacer oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive unit (MIRU) typing. The largest cluster of the TB isolates from Taipei was type MT11 (MIRU type 2233-2517-3533). A comparison of the MIRU type data for the Beijing strains from Taipei and previously published MIRU type data for the Beijing strains from Asian cities with major population of Chinese was analyzed. The six major Beijing MIRU types (MT01, MT02, MT08, MT11, MT21, and MT44) were found to be common in four Asian cities including Taipei, Singapore, Hong Kong, and Wuhan. Results of this study indicate that there is geographical difference in the distribution of different Beijing strains of MTB.     

Application of four molecular techniques for typing outbreak-associated Mycobacterium tuberculosis strains

We applied four molecular techniques for the typing of strains of Mycobacterium tuberculosis associated with outbreaks: RFLP of the IS6110 insertion sequence, spoligotyping, RAPD, and PCR-IS6110. All 4 techniques were applied to 18 strains which were shown by epidemiological data to be involved in 6 outbreaks. All the methods classified the strains into the same groups as the classical epidemiological data did, but RFLP of the IS6110 insertion sequence and spoligotyping are laborious techniques requiring more than a full day's work, whilst RAPD and PCR IS6110 are simple methods easily incorporated into the daily routine. Nevertheless, a large-scale process of standardization and evaluation is necessary in order to be able to establish the true value of the latter two methods as intraspecific characterization markers for M. tuberculosis isolates.     

A first insight into the genetic diversity and population structure of Mycobacterium tuberculosis in Zonguldak, Turkey.

This study evaluated the molecular epidemiology and biodiversity of Mycobacterium tuberculosis isolates in Zonguldak, Turkey, and investigated the presence and significance of the LAM7-TUR clone by spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) analysis. In total, 128 isolates were tested by spoligotyping; 25 selected isolates representative of the LAM7-TUR clone and similar types were also tested by MIRU-VNTR analysis. In total, 47 distinct patterns were revealed by spoligotyping, represented by 13 clusters containing between two and 28 isolates (94 isolates in total), and 34 unique patterns (a clustering rate of 73%). Using MIRU-VNTR analysis, the clustering relationships revealed by spoligotyping were confirmed. The most common spoligotyping profile was SIT53, followed by SIT41 (LAM7-TUR) and SIT50. The SIT284 clone was another phylogeographically specific clonal complex whose presence in Turkey may be endemic. The LAM7-TUR genotype was highly prevalent in Zonguldak.     

Investigation of Cyprinid herpesvirus-3 genetic diversity by a multi-locus variable number of tandem repeats analysis

Cyprinid herpesvirus-3 (CyHV-3), or koi herpesvirus (KHV), is responsible for high mortalities in aquaculture of both common carp (Cyprinus carpio carpio) and koi carp (Cyprinus carpio koi) worldwide. The complete genomes of three CyHV-3 isolates showed more than 99% of DNA sequence identity, with the majority of differences located in short tandem repeats, also called VNTR (variable number of tandem repeats). By targeting these variations, eight loci were selected for genotyping CyHV-3 by multiple locus VNTR analysis (MLVA). CyHV-3 strains obtained after sequential in vivo infections exhibited identical MLVA profiles, whereas samples originating from a single isolate passaged 6 and 82 times in vitro exhibited mutations in two of the eight loci, suggesting a relatively slow genetic evolution rate of the VNTRs. The method was subsequently applied on 38 samples collected in Indonesia, France and the Netherlands. Globally, the isolates grouped in two main genetic clusters, each one divided in two subgroups including either CyHV-3-U/I or CyHV3-J. Interestingly, Indonesian strains were rather distant from CyHV-3-J isolate. The results of the present study indicate that these VNTR molecular markers are efficient in estimating the genetic diversity among CyHV-3 isolates and are therefore suitable for further molecular epidemiological studies.