IS6110-限制性片段长度多态性DNA分型在结核分子流行病学研究中的应用

目的 建立宁夏、北京和上海等地结核分支杆菌发离株IS6110－RFLP DNA指纹图谱，观察其流行病学特征。方法 提取结核分支杆菌基因组DNA，经限制性内切酶PvuⅡ切割、琼脂糖凝胶电泳和Southern转印后，用荧光标记的IS6110DNA序列中245bp探针杂交，以核酸化学发光试剂盒探测荧光信号，比较各菌株指 IS6110拷贝数和带型，分析不同地理区域流行菌株的特点。结果 103例结核患的临床分离株，经245bp探针杂交后的指纹图谱显示大部分菌株含8－21个IS6110拷贝，在宁夏和北京地区流行的结核分支杆菌带型具有共同特点，并有成簇分布的现象，在上海分离株中发现1株零拷贝株和1株单拷贝株。结论 IS6110－RFLP DNA分型方法可用于我国流行的结核分支杆菌分子流行病学研究；宁夏分离株与北京分离株在基因上亲缘关系较相近。     

间隔区寡核苷酸分型技术用于结核分枝杆菌多重感染的初步研究

目的 初步评价间隔区寡核苷酸分型(Spoligotyping)技术在鉴定结核分枝杆菌多重感染中的应用。方法 选取结核分枝杆菌临床分离株,5 μm孔径滤膜过滤法制备单个细菌菌悬液,平板接种37 ℃培养,选取生长良好的单个菌落培养增菌,提取基因组DNA,Spoligotyping进行基因分型鉴定,比较临床分离株与相应的单克隆分离株的分型结果。结果 共选取32株结核分枝杆菌临床分离株,获得306个单克隆分离株。Spoligotyping分型鉴定结果显示30株结核分枝杆菌临床分离株与相应的单克隆分离株的分型结果一致,2株临床分离株的单克隆分离株呈现了多种基因型,多重感染率为6.25%。结论 Spoligotyping可用于快速,有效地区分结核分枝杆菌多重感染。     

联合应用表型分析、热休克蛋白65限制性片段长度多态性分析和测序鉴定养鱼水中的非结核分枝杆菌

目的了解养鱼水中非结核分枝杆菌（NTM）的分布情况。方法采集30份市售养鱼水标本，通过分离培养和生化反应初步鉴定，然后从培养菌落中提取DNA，PCR扩增65kD分枝杆菌抗原，扩增产物：（1）分别应用两种限制性内切酶BstE Ⅱ和Hae Ⅲ酶切，然后进行琼脂糖凝胶电泳和限制性片段长度多态性分析（PRA）；（2）直接测序。结果30份市售养鱼水中29份样本分枝杆菌培养阳性，其中6份样本分别生长出两种形态性状完全不同的菌落，共分离出35株分枝杆菌，表型特征均符合NTM。理化性质、PRA和测序鉴定发现，戈登分枝杆菌8株（23％）、龟-偶然分枝杆菌复合群8株（23％）、日内瓦分枝杆菌9株（26％）、不产色分枝杆菌1株，其余9株未鉴定到种。结论NTM广泛存在于市售养鱼水中，分子生物学方法与常规细菌学鉴定可互相补充，提高鉴定的正确性。     

1997-1998泰国的全国样本结核分枝杆菌限制性内切酶片段长度多态性研究

地点：1997—1998年间，作为全球项目的一部分，在泰国进行了全国性的抗结核药物耐药监测。 目的：评价IS6110杂交带谱和成簇的水平，由于样本收集时间较短，预计不会太高。 设计：共计828株分离株可做标准的IS6110杂交指纹分析。 结果：随着地理位置、病人年龄、对利福平和链霉素耐药的变化，限制性片段长度多态性也随之变化。北京株在青年病人中常见，离曼谷越远，其流行率也越低，但只有一个杂交带的分离株则正好相反。排除舍有5个以下拷贝IS6110的菌株，26．4％的菌株成簇。成簇在女性患者中更普遍。成簇菌株常常来自不同省份，如果有耐药，它们的耐药谱也不同。 结论：在泰国某些地区用IS6110杂交带谱的成簇性来推断近期传播的可靠性值得怀疑。在结核病高流行国家进行全国性的近期传播研究的评价应该谨慎。     

联合应用表型分析、热休克蛋白65限制性片段长度多态性分析和测序鉴定养鱼水中的非结核分枝杆菌

目的了解养鱼水中非结核分枝杆菌(NTM)的分布情况。方法采集30份市售养鱼水标本,通过分离培养和生化反应初步鉴定,然后从培养菌落中提取DNA,PCR扩增65kD分枝杆菌抗原,扩增产物:(1)分别应用两种限制性内切酶BstEⅡ和HaeⅢ酶切,然后进行琼脂糖凝胶电泳和限制性片段长度多态性分析(PRA);(2)直接测序。结果30份市售养鱼水中29份样本分枝杆菌培养阳性,其中6份样本分别生长出两种形态性状完全不同的菌落,共分离出35株分枝杆菌,表型特征均符合NTM。理化性质、PRA和测序鉴定发现,戈登分枝杆菌8株(23%)、龟-偶然分枝杆菌复合群8株(23%)、日内瓦分枝杆菌9株(26%)、不产色分枝杆菌1株,其余9株未鉴定到种。结论NTM广泛存在于市售养鱼水中,分子生物学方法与常规细菌学鉴定可互相补充,提高鉴定的正确性。     

多位点可变数量串联重复序列分析在北京家族结核分枝杆菌基因分型中的应用及位点筛选

目的对于北京家族菌株占绝大多数的感染人群,评价多位点可变数量串联重复序列（variable number tandem repeats,VNTR）分析（multiple loci VNTR analysis,MLVA）中不同位点组合在结核分枝杆菌（Mycobacterium tuberculosis,MTB）基因分型研究中的应用。并以IS6110限制性片段长度多态性（restriction fragment length polymorphism,RFLP）为参照,筛选有效位点。方法分别采用IS6110-RFLP、间隔区寡核苷酸分型（Spoligotyping）及MLVA不同位点组合对北京海淀区收集的MTB临床分离株进行基因分型研究,比较3种方法及MLVA不同位点组合的分型效果。结果 45株MTB分离株中86.7%为北京家族菌株,Spoligotyping和结核分枝杆菌散在分布重复单位（mycobacterial interpersed repetitive units,MIRU）-12系列的HGI（Hunter-Gaston Index）值分别为0.4313和0.8700,将45株MTB菌株分为10个和23个基因型。VNTR-9系列和IS6110-RFLP的分型结果一致,HGI值较高,为0.9980,将45株MTB菌株分为43个基因型。结论北京家族结核分枝杆菌在北京海淀区呈高水平流行。对于北京地区北京家族菌株占绝大多数的感染人群,VNTR-9系列MLVA是较为简便和高分辨率的分型方法 ,其分辨率能够达到MTB基因分型＂金标准＂IS6110-RFLP水平。     

结核分枝杆菌散在分布重复单位基因型分型法的应用研究

目的 建立结核分枝杆菌散在分布重复单位(mycobacterial interspersed repetitive units，MIRU)基因型分型法，评估该方法在我国应用的可行性。方法 采用简单数字表法随机抽取上海地区2000年至2002年保存的菌株，对其进行MIRU分型，并与间隔区寡核苷酸分型法(Spoligotyping)的结果进行比较。结果 用Spoligotyping方法对随机抽样的91株临床菌株进行基因型分型，得到20种基因型，其中81株(89％)属于北京基因型菌株，而用MIRU分型方法可将这些菌株分为46种基因型。81株北京基因型菌株可以分为39种不同的MIRU基因型。12个MIRU位点的多态性分析表明各位点有较大的区别，其中位点26显示了较高的多态性，位点16、31、40显示了中等程度的多态性。结论 MIRU分型方法简单快速，其数字化结果清晰可靠，是一种有效的结核分枝杆菌基因型分型方法。     

乙型肝炎病毒S基因限制性片段长度多态性分型方法的建立及应用

目的 建立乙型肝炎病毒(HBV)S基因片段聚合酶链反应-限制性片段长度多态性(RFLP)的基因分型方法并用于基因型与临床疾病谱关系的研究。 方法 比较GenBank中124株各基因型HBV全序列的S基因核苷酸序列及13株单独S基因序列,设计利用限制性内切酶Mbo Ⅰ、BsTN Ⅰ、BsmA Ⅰ、HpaⅡ酶切S基因片段鉴定HBV基因型(A-F)的方法。对贵州地区176份乙型肝炎患者血清进行基因分型并分析基因型与疾病谱的关系。直接测序2份B型和3份C型毒株的PCR扩增产物,以验证本酶切分型方法的正确性。 结果 测序结果证明本方法能够准确鉴定基因型。在176份标本中,B型100份(56.8％),C型76份(43.2％),未发现其他基因型。B型中无症状携带者(ASC)比例(40.0％)高于C型(15.8％),x2=12.16,P<0.005;慢性中度比例(14.0％)低于C型(31.6％),x2=7.88,P<0,005。 结论 本S基因片段PCR-RFLP基因分型方法简便、准确。贵州地区存在HBV B和C基因型。     

结核分枝杆菌北京基因型菌株大片段的多态性研究

目的 揭示结核分枝杆菌(Mycobacterium tuberculosis,MTB)北京基因型菌株的进化路径,在进化过程中产生的各个进化分支,以及每个分支的北京基因型菌株在人群中的流行情况。方法 收集2014年1月至2016年4月天津地区临床分离的567株MTB菌株,首先采用多重PCR试验分析菌株基因组中差异片段207(RD207)的缺失情况,以鉴定收集的菌株是否为北京基因型;然后分析所有的北京基因型MTB菌株基因组中差异区域RD105、RD181、RD150和RD142的缺失情况,以及菌株基因组NTF(noise transfer function)区中插入序列6110(IS6110)的存在情况。结果 567株临床分离的MTB菌株中,517株(91.2%)为北京基因型菌株。所有北京基因型菌株中,447株(86.5%)为NTF区含有IS6110的北京基因型现代株;70株(13.5%)为NTF区不含IS6110的北京基因型古代株。基于大片段的多态性分析,北京基因型菌株被分为5个亚型,其中RD181(+)的北京基因型菌株为22株(4.3%),且全部为北京基因型古代株。RD181(-)/RD150(+)和RD181(-)/RD150(-)的北京基因型古代株分别为41株(7.9%)和7株(1.4%)。447株现代菌株中,RD181(-)/RD150(+)和RD181(-)/RD150(-)的分别为404株(78.1%)和43株(8.3%)。结论 MTB北京基因型中的现代株是天津地区的主要流行株;北京基因型MTB在人群传播流行中已经进化出5个分支,其中RD181(-)/RD150(+)的北京基因型现代株为主要的流行分支。     

多重PCR和间隔区寡核苷酸分型技术应用于不同流行地区的牛结核病研究

目的 在牛结核病监测中联合应用菌株的多重PCR鉴定和间隔区寡核苷酸分型(spoligotyping),快速鉴定分离菌株并分析不同地区流行菌株的特点。方法 分别在牛结核病清净地区和流行地区进行牛型PPD变态反应试验,扑杀阳性牛后进行病理检查,并采集病料分离细菌。获取分离菌株,进行多重PCR鉴定和spoligotyping分型,分析不同地区的菌株特征。结果 结核病清净地区监测到16头牛型PPD阳性牛,扑杀后未见有病变,采集病料分离到4株分枝杆菌,经多重PCR鉴定均为非典型分枝杆菌。流行地区监测牛型PPD阳性23头,扑杀后发现14头有病变,采集病料后共有11头分离到疑似菌,经多重PCR鉴定均为牛型分枝杆菌。用spoligotyping分析共分为4个型,其中2个为未见报道的新型,报英国AHVLA牛结核spoligotyping数据库后获取通用编号SB1903和SB1904。结论 分离菌株中的优势菌株为首次报道的SB1903,但也检出有国际流行菌株SB0140,证实该地区牛结核病的流行是以“独特菌型地域内相互传播为主、输入性感染为辅”为特征。本次监测发现国内有SB0140菌株分布,提示应调查该型菌是否已经传染至我国人间。     

HLA-DR: Serology versus Restriction Fragment Length Polymorphism

The results of the serological typing and the patterns of restriction fragment length polymorphisms for the detection of HLA-DR gene products are compared. The data shown demonstrate that the use of DNA typing gives a clearer definition of the HLA-DR antigens and that the HLA-DR polymorphism is greater than detected by serology.     

不同宿主肝片吸虫DNA限制性片段长度多态性分析

本文首次对分离自黄牛、水牛和牦牛的肝片吸虫的基因组DNA，以BamHⅠ、BglⅡ、EcoRⅠ、HindⅢ和PStⅠ等6种限制性内切酶消化后进行琼脂糖电泳，分析比较它们基因组DNA的限制性片段长度多态性（RestrictionFragmentLengthPolymorphism，RFLP）；同时用地高车标记的牦牛肝片吸虫基因组DNA为探针对不同宿主肝片吸虫DNA进行Southern印迹杂交，限制性内切酸酶切结果显示只有两种限制性内切酶（BamHⅠ和EcoRⅠ）在寄生于牦牛的肝片吸虫中检测到多态性，而水牛肝片吸虫与黄牛肝片吸虫基因组DNA之间没有检测到多态性，根据此结果计算了牦牛肝片吸虫与黄牛肝片吸虫和水牛肝片吸虫基因型间的遗传距离，Southern杂交结果显示牦牛肝片吸虫基因DNA的杂交谱带与黄牛肝片吸虫、水牛肝片吸虫基因组DNA的杂交港带存在一定的差异，本文的实验结果表明牦牛肝片吸虫与黄牛肝片吸虫、水牛肝片吸虫的亲缘关系相对较远，而水牛肝片吸虫与黄牛肝片吸虫的亲缘关系较近。     

Identification and evolution of an IS6110 low-copy-number Mycobacterium tuberculosis cluster

A cohort of 56 patients infected with related strains of Mycobacterium tuberculosis, the S75 group, was identified in a New Jersey population-based study of all isolates with a low number of copies of the insertion element IS6110. Genotyping was combined with surveillance data to identify the S75 group and to elucidate its recent evolution. The S75 group had similar demographic and geographic characteristics. Seventeen persons (30%) were linked epidemiologically. The S75 group was segregated from other low-copy-number isolates on the basis of several independent molecular methods. This group included 3 IS6110 genotype variants: BE, H6, and C28, containing 1, 2, and 3 IS6110 insertions, respectively. IS6110 insertion site mapping and comparative sequence analysis strongly suggest a stepwise acquisition of IS6110 elements from BE to H6 to C28. S75 represents a locally produced strain cluster that has recently evolved. The combination of multiple molecular tools with traditional epidemiology provides novel insights into dissemination, local transmission, and evolution of M. tuberculosis.     

Restriction Fragment Length Polymorphism in the Adhesin Gene hpa A of Helicobacter pylori

Objectives : To assess the degree of restriction fragment length polymorphism (RFLP) in the Helicobacter pylori adhcsin gene hpa A and to determine the molecular basis of RFLP in this gene. Methods : A 375-bp, poly-merase chain reaction-amplified internal sequence of hpa A obtained from SO different H. pylori isolates, was restricted with Sau 3A and Hin fl, individually. Poly-merase chain reaction products representing different RELP types were sequenced. Results : Seven different polymorphic types were found in hpa A. Base substitutions at only four positions, two in Sau 3A and two in Hin fl sites, account for all of the RFLP types, including the size of the restriction fragments determined by gel electrophoresis. Most, 90%, of the base substitutions are very conservative, i.e. , either do not change the encoded umino acid or substitute a homologous amino acid, and cause no detectable antigenic or functional effect on hpa A The region of hpa A encoding the receptor-binding motif was particularly well conserved. Conclusions : RFLP typing of hpa A using Sau 3A and Hin fl provides an additional tool for comparing the genetic relatedness of H. pylori isolates collected during epidemiological and/or treatment studies.     

IS6110-RFLP and spoligotyping of Mycobacterium tuberculosis isolates in Iran

To evaluate and compare the usefulness of IS6110-restriction fragment length polymorphism (RFLP) and spoligotyping in the epidemiology of tuberculosis in Iran, Mycobacterium tuberculosis complex strains, isolated in 2 different areas of Iran, were subjected to RFLP and spoligotyping. The average number of IS6110 copies per strain was 11 and ranged from 5 to 18 among the M. tuberculosis strains. In total, among the 62 isolates, 56 different patterns were observed. 50 strains had unique RFLP patterns (89%) and 12 (11%) revealed patterns that were found among at least 1 other isolate. Spoligotyping of 97 isolates resulted in 42 different patterns, of which 72% were found in 15 clusters. 14 (29%) out of 48 investigated isolates were resistant to 1 or more antituberculosis drugs and 57% of the resistant isolates were isolated from Afghan immigrants. Ten percent of the isolates represented the Beijing genotype, including 4 of the 14 (36%) resistant strains. Three of these resistant Beijing strains were isolated from Afghan patients. IS6110-RFLP typing could be useful for studying the epidemiology of tuberculosis in Iran. IS6110 patterns were polymorphic and the average IS6110 copy number was high.     

Detection of Mycobacterium tuberculosis in clinical samples using insertion sequences IS6110 and IS990

To detect Mycobacterium tuberculosis in clinical samples, we used the M. tuberculosis-complex specific insertion sequence IS990 as the target in a simple DIG-PCR ELISA assay, as this element is present as a single copy in all strains of M. tuberculosis we have examined to date. The IS990 test was compared with a similar PCR that utilizes IS6110 as target. For detection of PCR product, digoxigenin-11-dUTP (DIG-dUTP) was incorporated into the product. After amplification, the PCR product was hybridized with biotinylated capture probe, which was complementary to the inner part of the amplicon. The hybrid was captured onto streptavidin-coated microtiter plate and DIG-labeled PCR product was detected using a peroxidase-conjugated antibody to DIG. We evaluated DIG-PCR ELISA for the detection of M. tuberculosis DNA in 265 respiratory and non-respiratory specimens taken from patients with known and suspected tuberculosis disease or from controls. The sensitivity and specificity of both IS990-based test and IS6110-based test was 96.5% and 95.3% respectively, comparable to the sensitivity and specificity of the IS6110-based test. The results demonstrate that the IS990 PCR ELISA test is a rapid and sensitive tool for the detection and identification of M. tuberculosis in clinical samples, and may have advantages to the more widely used IS6110-based tests, particularly in areas where IS6110-negative strains are found.     

结核分枝杆菌IS6110探针的克隆化

目的　制备克隆化结核分枝杆菌IS6110探针。方法 将IS6110的PCR扩增245bp片段克隆至质粒载体pCR2.1中。结果 得到了含有IS6110克隆化245bp的质粒菌株。结论 克隆化的探针易操作且一致性较好。     

The role of IS6110 in the evolution of Mycobacterium tuberculosis

Members of the Mycobacterium tuberculosis complex contain the transposable element IS6110 which, due to its high numerical and positional polymorphism, has become a widely used marker in epidemiological studies. Here, we review the evidence that IS6110 is not simply a passive or 'junk' DNA sequence, but that, through its transposable activity, it is able to generate genotypic variation that translates into strain-specific phenotypic variation. We also speculate on the role that this variation has played in the evolution of M. tuberculosis and conclude that the presence of a moderate IS6110 copy number within the genome may provide the pathogen with a selective advantage that has aided its virulence.