IS6110限制性片段多态性分析标准方法的建立及其在结核分枝杆菌分子分型中的应用

目的 建立IS6110限制性片段多态性分析(IS6110-RFLP)标准方法 并评价该方法 的分型能力.方法 采用核酸提取、PCR、限制性内切酶分析、Southern杂交、琼脂糖凝胶电泳等技术,结合Gel-Pro analyzer 3.1和BioNumeries(Version 5.0)软件,对78株结核分枝杆菌插入序列IS6110-RFLP进行分析.结果 确定标准化的IS6110-RFLP技术,包括核酸提取、PCR、限制性内切酶分析、Southern杂交、琼脂糖凝胶电泳等实验步骤及标化参数的相关数据分析软件的使用;采用该技术,将78株结核分枝杆菌分为75个不同的基因型,分别归属于11个基因簇,其中有52株归属于同一个基因簇,占菌株总数的66.7%(52/78).结论 建立标准化的IS6110-RFLP技术方案,该方法 具有很强的基因分型和株水平鉴定能力,可用于结核病的病原学监测.     

三种不同的DNA分型技术在158株结核杆菌研究中的应用

目的评价IS6110限制性片段长度多态性(RFLP)、间隔区寡核苷酸分型(Spoligotyping)及分枝杆菌散在重复单位(MIRU)三种分型方法在结核病流行病学研究中的应用。方法对158株结核分枝杆菌临床分离株应用IS6110RFLP、Spoligotyping及MIRU三种分型方法进行鉴定。结果应用三种分型方法产生的类型数分别为118、20和105个。IS6110RFLP的分辨率大于Spoligotyping,MIRU的分辨能力与IS6110RFLP接近。在MIRU的12个区中,重复区4、10、26、40具有较高的多态性。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。结论应用IS6110RFLP、Spoligotyping及MIRU三种分型方法进行结核病流行病学研究具有重要意义且非常有效,可以发现中国不同地区菌株的不同特点。     

用IS6110限制性片段长度多态性方法分析中国南方部队结核分支杆菌DNA指纹特征

目的：分析南方部队结核病患者和当地患者中结核分支杆菌分离株DNA指纹特征，探讨南方部队结核病的分子流行病学特征。方法：用限制性内切酶PvuⅡ消化结核分支杆菌DNA，后用琼脂糖凝胶电泳，再用Southern免疫转印，用[α^32P]-dCTP标记的DNA IS6110序列中的245bp片段作探针，进行杂交后得到限制性片段长度多态性图谱，结合一般流行病学资料加以分析比较。结果：共检测185株结核分支杆菌分离株。检测菌株的IS6110拷贝数范围为1～22。部队患者和当地患者的IS6110拷贝数分布差异无显著姓。部队患者结核菌分离株的IS6110拷贝数主要集中在6～20个，当地分离株主要集中在7～20个；全部菌株指纹特征分成8个组，部队分离株和当地分离株均主要集中在Ⅰ、Ⅱ、Ⅲ 3个组里。耐药菌株指纹特征在各组中的分布与敏感菌株差异有显著性；患者是否接种卡介苗在各组中的分布差异无显著性。结论：南方部队患者与当地患者结核菌分离株在遗传关系上较接近，在基因水平上相关程度较强。提示部队结核病的发生与当地结核分支杆菌菌株的传播密切相关。     

某区2004年中、小学生结核病普查

目的：评估本地区中小学生结核病流行病学状况。方法：用传统流行病学和分子流行病学方法。对125893名中、小学生进行PPD实验和X线检查；结核病人进行痰结核菌涂片和培养；对结核分枝杆菌分离株进行分子流行病学研究，用标准RFLP技术使用IS6110插入序列对结核分枝杆菌进行分型和成簇分析。结果：125893名中、小学生中（95．9％）接种过BCG。小学生1～4年级PPD阳性20963例，阳性率47．9％，其中强阳性17例；确定诊断结核感染3例、颈淋巴结结核4例，原发综合征和肺门淋巴结结核10例。小学5、6年级和中学生共X线异常阴影并确诊为肺结核48例。58例肺结核病人痰抗酸染色阳性15例（25．9％）、培养阳性42例（72．4％）；患病率46．1／10万；涂阳患病率13．5／10万。42株结核分枝杆菌中，IS6110 RFLP分析23株（55％）成簇，拷贝相似性高；其余19株IS6110拷贝呈现多态性。最大簇包含3例病人。结论：本地区中、小学生结核病患病率较低；新近传播病例未形成较大成簇，近期可能无集团感染和暴发发生。     

以PCR为基础的分型方法在结核病研究中的应用

1、以RFLP为基础的分型方法 1．1限制性片段长度多态性分析（restriction fragment length polymorphism，RFLP） 它是一种最早使用的基因分型方法，该方法是针对结核分支杆菌基因组DNA上的特征片段，如插入序列IS6110、IS1081，多态性富含GC重复序列，（GTG）5寡聚脱氧核苷酸等，利用限制性内切酶酶切特征性片段上的某一位点，电泳分离，而形成结核分支杆菌DNA指纹图谱。     

结核杆菌实验室甲醛终末消毒效果辅助评价方法建立

目的 建立一种便捷可靠的辅助评价结核杆菌实验室终末消毒效果的新方法。方法 首先以结核分枝杆菌复合群特异性插入序列IS6110为靶标自主设计引物IS6110P,并经结核分枝杆菌标准株H37Rv的培养物验证其特异性和灵敏度;其次,采用H37Rv培养物抽滤膜经甲醛终末消毒后分别采用普通核酸提取法和完整基因组提取核酸法提取核酸,再经IS6110P引物进行特异性PCR扩增验证其辅助评价甲醛终末消毒效果的情况,同时对抽滤膜进行分离培养以验证其评价结果;最后,采集经甲醛终末消毒结核杆菌实验室内的拭子和大气样本采用上述方法检测,并与分离培养结果相验证,以检测该方法的评价效果。结果 IS6110P可灵敏特异地对结核分枝杆菌进行扩增,检测限可达3.5 cfu/R;H37Rv培养物抽滤膜甲醛终末消毒后经普通核酸提取扩增仍有特异性核酸片段检出,而经完整基因组提取核酸扩增无特异性核酸片段检出;终末消毒后的结核分枝杆菌实验室采用普通核酸提取仍可扩增出结核分枝杆菌特异性核酸片段,而完整基因组提取核酸无特异性核酸扩增片段检出;平行的结核分枝杆菌分离培养实验结果均为阴性。结论 采用IS6110P扩增结核分枝杆菌完整基因组的核酸提取物为辅助评价结核分枝杆菌实验室终末消毒效果的灵敏、便捷和可靠的新方法。     

浙江省宁波地区结核分枝杆菌VNTR基因分型研究

目的初步探讨数目可变串联重复序列(VNTR)技术在宁波地区结核分枝杆菌基因分型中的应用。方法根据文献选取7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,分析结核分枝杆菌DNA多态性。结果本研究组共对138株结核分枝杆菌的7个VNTR位点进行了检测,共产生7个基因簇和123个独立基因型,成簇率为5.80%,VNTR-7位点基因分型技术分辨率指数(HGI)为0.999 1。结论VNTR-7位点基因分型技术对宁波地区结核分枝杆菌的分析有较高的分辨力,操作简便,适用于宁波地区结核病分子流行病学研究。     

异烟肼诱导致单耐异烟肼结核分枝杆菌假定药物外排泵基因表达量变化的初步研究

目的 <\b>检测单耐异烟肼结核分枝杆菌经异烟肼诱导培养前后假定药物外排泵基因表达量的变化,探讨可能导致结核分枝杆菌对异烟胼耐药的外排泵基因及导致基因高表达的原冈.方法 <\b>选取35株单耐异烟肼结核分枝杆菌临床分离株、10株全敏结核分枝杆菌临床分离株和H37Rv(标准对照),对各菌株进行无异烟肼和亚抑菌异烟肼浓度(1/4 MIC)的7H9液体培养基诱导培养,提取RNA后反转录并采用real-time PCR技术分别检测27个假定外排泵基因表达量的变化,用公式2-ΔΔCT计算每个假定基因相对表达量的差异倍数.结果 <\b>耐药株中27个假定外排泵基因有13个基因高表达,高表达Rv1258c基因的菌株数最多为6株,其次是高表达Rv2265和Rv0849的菌株各有5株.35株菌中14株菌(40.00％)有高表达的外排泵基因,6株(17.14％)菌株均只有1个假定外排泵基因高表达；8株(22.86％)菌株有两种及以上基因高表达,其中4、2、1、l株菌株分别有2、4、5、7个基因高表达；10株全敏菌株和H37Rv 27个假定外排泵基因均无高表达.结论 <\b>Rv1258e、Rv2265和Rv0849等基因可能是导致结核分枝杆菌对异烟肼耐药的外排泵基因,异烟肼可能为结核分枝杆菌部分假定外排泵基因的诱导物而导致其激活并高表达.     

中国北方部分地区北京谱系结核分枝杆菌群体遗传关系的分析

目的 <\b>应用分子遗传学方法探讨234株北京谱系结核分枝杆菌的起源进化及群体间基因流动特征.方法 <\b>对来源于中国北方五省区(黑龙汀、吉林、辽宁、内蒙古和宁夏)234株北京谱系结核分枝杆菌进行24位点可变数目串联重复序列(VNTR)基因分型,计算各VNTR位点的等位基因多态性(h);从个体水平分析其系统发育,即构建NJ树和最小生成树;构建群体水平系统发育树,并在此基础上通过贝叶斯模型计算最近共祖年代;通过分子方差分析了解五省区菌株间的基因流动情况.结果 <\b>24个VNTR位点的等位基因多态性较低(h值:0.000 ～ 0.744).234株北京谱系结核分枝杆菌分散存在NJ树各个进化分支,最小生成树中约62.0％(145/234)的菌株被划分到同一“克隆复合群”.群体水平系统发育树显示234株菌与MIRU-VNTRplus数据库(http://www.miru-vntrplus.org)中的北京谱系结核分枝杆菌的遗传关系最近(Bootstrap值为100);最近共祖年代为5308年(95％CI:4263 ～ 6470年).通过分子方差分析发现吉林与黑龙江、辽宁菌株之间,内蒙古与宁夏菌株之间遗传分化系数的差异无统计学意义(P＞0.05).结论 <\b>中国北方五省区北京谱系结核分枝杆菌的遗传相似性较高,菌株间种系发生关系不明显.推测这些菌株由近期(约5000年前)结核分枝杆菌北京谱系的某一“克隆”进化而来.地理位置接近的区域,菌株间存在基因流动现象.     

结核分枝杆菌多位点可变数目串联重复序列分型方法标准化操作程序的建立

目的 建立结核分枝杆菌多位点可变数目串联重复序列分析(MLVA)的标准化方法,评价该方法的分型能力、应用价值.方法 选取15个位点,采用核酸提取、PCR和琼脂糖凝胶电泳等技术,结合BioNumeries(Version 5.0)软件,对中国54株结核分枝杆菌临床分离株进行分型.结果确定标准化的MLVA方法,包括细菌分离培养、核酸提取、PCR、琼脂糖凝胶电泳等实验步骤及标化参数的相关数据分析软件的使用.VNTR位点确定为ETRA、ETRB、ETRC、ETRD、ETRE、MIRU10、MIRU16、MIRU23、MIRU26、MIRU27、MIRU39、MIRU40、Mtub21、Mtub30和Mtub39.结论建立MLVA标准化技术方案,该方法操作简便,分型鉴定能力及实验室间结果可比性强,便于网络化,有利于结核病流行中追溯传染源,分析流行趋势.     

Utility of gene analysis for evaluation of the current status of tuberculosis developing long after primary infection

PURPOSE: The purpose of this study was to examine the utility of gene analysis of Mycobacterium tuberculosis as a means of determining the current status of tuberculosis developing in patients long after primary infection and identifying the source of infection. METHODS: We analyzed two outbreaks of tuberculosis involving chronic carriers in 2004 within Okayama prefecture. DNA was extracted from Mycobacterium tuberculosis stains isolated from the patients, and subjected to IS6110-RFLP and PGRS-RFLP analyses. The resulting IS6110-RFLP patterns were compared with a database (compiled since December 1999) of RFLP patterns of isolates from newly registered tuberculosis patients in the prefecture. Mutations in the rpoB gene for rifampicin resistance and the katG gene for isoniazid resistance ofMycobacterium tuberculosis were detected by real-time PCR followed by melting curve analysis. Bacterial isolates were tested for antimycobacterial susceptibility by the microdilution susceptibility method and the proportion method on Ogawa's medium. RESULTS: In outbreak 1, an epidemiological survey suggested that two individuals contracted tuberculosis from the same patient after an interval of about 20 years. They differed from the suspected patient in the drug susceptibility patterns of bacterial isolates and mutations in the drug susceptibility-related genes, but the results of IS6110-RFLP and PGRS-RFLP analyses supported a conclusion of a common source of infection. In outbreak 2, a hospital employee developed primary multidrug-resistant tuberculosis of unknown origin. Comparison between the IS6110-RFLP patterns of the employee's bacterial isolate and the IS6110-RFLP database showed identity to a bacterial isolate from a chronic carrier who had died in the same hospital 4 years and 8 months earlier. Moreover, the two isolates were identical regarding drug susceptibility patterns and mutations in drug resistance-related genes, suggesting a nosocomial infection. CONCLUSION: These results indicates that tuberculosis gene analysis provides useful information for comprehension of the current status of tuberculosis developing in patients long after primary infection. Genotype database construction may allow detection of the source of infection, which cannot be identified by contact examination alone.     

Role of spoligotyping and IS6110-RFLP in assessing genetic diversity of Mycobacterium tuberculosis in India

In the present study, genetic diversity analysis of Mycobacterium tuberculosis isolated from patients attending a tertiary care hospital, North India, has been attempted. Eighty three isolates of M. tuberculosis were subjected to DNA fingerprinting using spoligotyping and IS6110-RFLP techniques. Spoligotype patterns showed that central Asian (32.5%), ill defined T (13.2%) and Beijing (10.8%) families were predominant in ongoing transmission of the bacterium. Two STs; ST26 (CAS_Delhi) and ST1 (Beijing) represented 36.1% of the total M. tuberculosis population in eastern Uttar Pradesh, North India. IS6110 RFLP analysis showed that isolates having low and zero copy number of the IS element were 15.6% and 19.2%, respectively. Out of the 47 isolates clustered by spoligotyping, 40 could be further differentiated as unique strains by IS6110-RFLP. Therefore, this study recommends that both the techniques be used simultaneously for DNA fingerprinting of M. tuberculosis in India.     

华东农村地区利福平耐药结核病的 分子流行病学特征研究

目的 描述华东农村地区利福平耐药结核分枝杆菌(结核菌)的耐药相关分子特征和成簇性规律.方法 以浙江省和江苏省两个农业县结核病防治所一年内所有登记的结核菌检测阳性肺结核病例为研究对象,收集痰标本、培养分离菌株,并进行问卷调查;对培养获得的结核菌株采用比例法进行药敏试验.对获得的65株利福平耐药结核菌株进行rpoB基因利福平耐药决定区和katG基因异烟肼耐药相关热点区测序,采用间隔区寡核苷酸分型(spoligotyping)方法识别北京株,采用IS6110限制性内切酶多态指纹技术(RFLP)对分离株进行基因分型和成簇性分析.结果 92%(60/65)的利福平耐药菌株在利福平耐药决定区531位(57%)、526位(29%)或516位(11%)发生突变,82%(49/60)的rpoB基因突变菌株为耐多药结核菌(MDR-TB);spoligotyping识别H{54株(83%)北京家族菌株;IS6110-RFLP发现共有24株(37%)利福平耐药菌株成簇,形成11个簇,提示簇中病例的结核菌近期传播.所有成簇菌株都为MDR-TB.共有7个簇,簇内菌株具有不完全相同的利福平耐药相关的rpoB基因突变.分析可能与成簇性相关的因素,初治病例在菌株成簇患者中的比例高于在菌株非成簇患者中所占的比例(初治/复治:OR=3.342,95%CI:1.081～10.32).结论 在其他耐药结核菌(如异烟肼耐药结核菌)中进一步产生对利福平的选择性耐药和生长,很有可能是利福平耐药结核病流行的基础,而既往较长的不规则治疗史也为利福平耐药的发生和传播提供条件.     

Interpreting genotype cluster sizes of Mycobacterium tuberculosis isolates typed with IS6110 and spoligotyping

Molecular techniques such as IS6110-RFLP typing and spacer oligonucleotide typing (spoligotyping) have aided in understanding the transmission patterns of Mycobacterium tuberculosis. The degree of clustering of isolates on the basis of genotypes is informative of the extent of transmission in a given geographic area. We analyzed 130 published data sets of M. tuberculosis isolates, each representing a sample of bacterial isolates from a specific geographic region, typed with either or both of the IS6110-RFLP and spoligotyping methods. We explored common features and differences among these samples. Using population models, we found that the presence of large clusters (typically associated with recent transmission) as well as a large number of singletons (genotypes found exactly once in the data set) is consistent with an expanding infectious population. We also estimated the mutation rate of spoligotype patterns relative to IS6110 patterns and found the former rate to be about 10-26% of the latter. This study illustrates the utility of examining the full distribution of genotype cluster sizes from a given region, in the light of population genetic models.     

13个VNTR位点用于113株结核分支杆菌的基因分型研究

目的 应用数目可变串联重复序列（VNTR）分子分型技术，对北京地区113株肺结核临床分离菌株进行分型研究，探讨北京地区菌株DNA多态性和基因型特征。方法 采用PCR和琼脂糖凝胶电泳技术对结核分支杆菌13个VNTR位点进行检测，并应用Gel-Pro analyzer 3．1软件和BioNumerics3．0软件进行结果分析。结果 113株结核分支杆菌可分为4个基因型（分别为Ⅰ、Ⅱ、Ⅳ和Ⅴ型），其中Ⅰ型占92．0％（104/113），其他3型所占比例很小，分别为Ⅱ型占4．4％（5／113），Ⅳ和Ⅴ型均为1．8％（2／113），而标准菌株H37Rv在分型中为独立的一个基因型，即Ⅲ型。结论 北京地区的结核分支杆菌存在基因多态性，其主要流行型为Ⅰ型。     

DRE-PCR (Double Repetitive Element-Polymerase Chain Reaction)--a new molecular-epidemiologic method in the detection of tuberculosis

In a pilot study Double Repetitive Element-Polymerase Chain Reaction 20 clinical isolates of Mycobacterium tuberculosis from Guatemala and 49 strains from Prague were typed. This technique is based on direct evidence of repetitive elements IS6110 or PGRS and does not require DNA purification, digestion by endonuclease nor Southern blot hybridization. Preliminary examination of Guatemalian strains revealed a striking identity or similarity of DRE-PCR profiles while the Prague strains were characterized by conspicuous polymorphism. The Prague strains were examined in a total number of 13 series of electrophoreograms and subsequently subjected to automated analysis with GelCompar software. The DRE-PCR method is suitable for screening of a major number of clinical isolates of M. tuberculosis in laboratories equipped with a minimum of technical facilities for the PCR method, furthermore it requires facilities for synthesis of the necessary primers and at least basic knowledge of molecular biology.     

Molecular epidemiology of tuberculosis in the Czech Republic, 2004: analysis of M. tuberculosis complex isolates originating from the city of prague, south Moravia and the Moravian-Silesian region

OBJECTIVES: To compare M. tuberculosis complex genotypes from representative regions of the Czech Republic in order to estimate changes in strain prevalence and in the extent of imported drug-resistant tuberculosis. METHODS: Primary M. tuberculosis complex isolates (n=155) and follow-up isolates (n=15) from 155 patients from the first half of 2004 (98 from Prague, 37 from South Moravia and 35 from the Moravian-Silesian region) were genotyped by IS6110-RFLP, spoligotyping, and partly by VNTR-genotyping. RESULTS: Based on IS6110-RFLP, 110 of 155 (71%) primary isolates were unique. Forty-five isolates (29%) were found in 15 clusters comprising two to six patients and all but one cluster were also discriminated by MIRU-VNTR-genotyping. Four clusters comprised patients from different regions, and six were ongoing for several years. An indication of MDR-strain transmission was found in one instance. All four Beijing-type isolates with any resistance were associated with immigration from Eastern Europe. CONCLUSIONS: The molecular epidemiological data of this period-prevalence, population based study and its comparison to earlier investigations point to a low extent of clustering between M. tuberculosis complex isolates in representative regions of the Czech Republic. Few clusters extending geographically and/or over several years were identified, providing a means for an in-depth analysis of risk factors of transmission. Beijing genotype isolates were shown to increase in prevalence to reach 6.5%. Drug resistant isolates of this genotype were associated with immigration of from Eastern Europe, although direct transmission of a resistant isolate was probable only in one of eleven cases.