吉林市结核分支杆菌IS6110 DNA指纹图谱特征分析

目的　分析吉林市结核分支杆菌IS6110 RFLP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IS6110 RFLP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IS6110 RFLP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)IS6110 RFLP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。     

北京结核菌株分子流行病学的研究

目的：北京结核分枝杆菌分子流行病学的方法。方法（1）使用标准化的RFLP法检测并分析从128位初治患者分离的结核分支杆菌IS6110指纹多态性;（2）对选例患者进行社会人口学和流行病学调查问卷;（3）分析结核分枝杆菌IS6110指纹多态特征与流行病学的关系。结果（1）根据IS6110指纹特征的同源性,检测的128株结核分枝杆菌可基本分为3组,A、B组的菌株间因具有较高的同源性而被称为：北京基因型”菌株,并且此类菌株在检测菌株中所占的比例较高（103／128,80．5％）;C组菌株间同源性较低,多态性变化多而被称为“非北京基因型”,仅占19．5％。（2）从较低年龄（＜40岁）,患者分离菌株的多态性变化,与从较高年龄（＞40岁）患者分离菌株多态性变化比较存在差异,提示“北京基因型”菌株不是由于BCG接种而造成的选择性优势。（3）检测菌株的多态性变化与患者结核病接触史、患者长期居住地、菌株药物敏感性情况无明显联系。结论：结核分枝杆菌的指纹分析对结核病现代流行病学的研究是一个有用的工具。     

延边州结核分枝杆菌DNA指纹图谱特征分析

目的　建立延边州结核分枝杆菌IS6110 RFLP指纹图谱 ,并分析朝鲜、汉两族的图谱特征。方法 97株结核分枝杆菌分离株 ,提取DNA ,经酶切 ,电泳 ,Southem转印 ,探针杂交 ,发光自显影技术得到IS6110 RFLP图谱;按其指纹图谱特征的同源性高低进行分析。结果 延边州结核分枝杆菌IS6110拷贝数目平均为 13个;57.7%菌株可成簇 ,具有“北京基因型”特征;IS6110 RFLP图谱特征分布无地域差异 ,无民族差异。结论 延边州结核分枝杆菌分离株同北京分离株有较近的亲缘关系 ,但其多态性较高。     

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IS6 110DNA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VanEmbden推荐的结核分支杆菌DNA指纹标准方法 ,构建标准菌株Mt14 32 3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IS6 110DNA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Gelcompar 4 .1(AppliedMaths ,Kortrijk ,Belgium )软件对上述菌株的指纹图谱进行聚类分析。结果 Mt14 32 3标准菌株和 74例临床分离菌株IS6 110DNA指纹图谱结果与国外同类报道一致 ;其中 2 4 .3% (18/74 )的结核菌株的IS6 110DNA指纹相似值在 1～ 0 .6 5之间 ,鉴定结果为它们均是北京家族结核分支杆菌 ;IS6 110 0 1个拷贝和 2个拷贝菌株较多 (2 1/74 )。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。     

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。     

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。     

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。     

结核分支杆菌DNA指纹技术及其应用研究

目的：探讨北京及其周围地区结核分杆菌的分布特征。方法：构建以IS6110为基础的结构分支杆菌DNA指纹图谱,应用Microbiology Information Numeral Taxonomy System软件进行处理,并且x2检验比较不同组别结核病人临床分离菌株成族（clustering)率的差别,结果：H37Rv,BCG两个标准菌株和62例临床分离菌株IS6110DNA指纹结果与国外同类报道一致,其中70％（44／62）的临床分离菌株IS6110DNA指纹相似值在1－065之间;分组统计结果显示;男性组与女性组成族率之间存在显著性差异（P＜0．05）。结论：北京及周围地区结核分支杆菌临床分离菌株多数遗传关系较近,在基因水平上相关程度较强。结核分支杆菌在男性人群中的传播频率可能较女性更高。     

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。     

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。     

Molecular epidemiology of Mycobacterium tuberculosis in China: a nationwide random survey in 2000.

OBJECTIVE: To determine the contribution of the Mycobacterium tuberculosis (TB) Beijing genotype to the population structure of M. tuberculosis complex in China. DESIGN: Genotypes of 441 mycobacterial isolates were determined by spoligotyping and IS6110 restriction fragment length polymorphism (RFLP). The isolates were from a nationwide random survey for the epidemiology of TB conducted in 2000, and the drug susceptibility patterns and epidemiological data were known. RESULT: A total of 408 samples contained M. tuberculosis as determined by spoligotyping. Of the M. tuberculosis strains, 64.9% (265/408) were of the Beijing genotype. Using the chi-squared test, no statistically significant differences were observed in the proportion of Beijing genotype TB in patients of different sex, age or living in different areas of the country, but a significant difference was observed with multidrug-resistant (MDR) TB: 77.8% (42/ 54) of MDR isolates were of the Beijing genotype vs. 60.2% (213/354) of the drug-susceptible isolates. In the multivariate logistic regression model, the Beijing genotype was significantly associated with region and not with MDR. CONCLUSION: The M. tuberculosis Beijing genotype has prevailed in China for at least five decades and is associated with region.     

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.     

Epidemiological evidence of the spread of a Mycobacterium tuberculosis strain of the Beijing genotype on Gran Canaria Island.

Molecular epidemiological studies suggest that particular Mycobacterium tuberculosis strains have an enhanced capacity to spread within a community. One strain, the Beijing genotype, has been associated with outbreaks in a number of communities throughout the world. IS6110 restriction fragment length polymorphism (RFLP) analysis was performed on M. tuberculosis isolates from 566 of the 721 patients (78.5%) diagnosed with tuberculosis (TB) on Gran Canaria Island from 1993 to 1996, as well as 35% of isolates from 1991-1992 (85 strains). RFLP identification of the family of strains of the Beijing genotype was confirmed by spoligotyping. Medical records of all patients were reviewed and epidemiological links were identified. Of 566 M. tuberculosis isolates from 1993 to 1996 with RFLP available, 72% belonged to clusters. The largest contained 75 cases and was caused by a strain of the Beijing genotype that was introduced to the island in 1993. It was found in 10 patients in 1993 (5.5%), 12 in 1994 (8.1%), 18 in 1995 (16.4%), and 35 in 1996 (27.1%). Epidemiological linkage was confirmed for 68% of cases. This study has demonstrated rapid dissemination of this strain of the Beijing genotype. This genotype might play an important role in the future of the worldwide tuberculosis epidemic.     

Molecular genetic characteristics of Mycobacterium tuberculosis isolated from patients operated on for pulmonary tuberculosis

The RFLP-IS6110 assay was used to genotype 67 Mycobacterium tuberculosis isolated from different specimens (including intraoperative ones) of 24 patients operated on for chronic progressive pulmonary tuberculosis who lived in north-western Russia. More than half (53%) of 17 types of RFLP profiles of isolates are identical and typical of M. tuberculosis of the Beijing family. Comparing the results of isolate genotyping with clinical, microbiological, and pathomorphological findings allows cases of endogenous reactivation to be differentiated from exogenous (nosocomial, in particular) reinfection in recurrence. Beijing genotype strains are shown to cause more severe unarrested course of drug-resistant fibrocavernous tuberculosis. At the same time such strains are more frequently detected in relatively young persons with a shorter duration of the disease, which reflects current clonal Beijing genotype dissemination that present a severe epidemiological hazard.     

贵州某结核病高发县结核分枝杆菌流行情况的初步研究

目的分析结核病高发县结核分枝杆菌临床分离株及其基因型特征,探索高疫情地区结核菌株流行情况。方法在罗甸县结核病定点医院门诊收集临床分离的分枝杆菌菌株,用PNB生长试验、结核分枝杆菌散在重复单位(VNTR) 和RD105缺失基因检测法分别进行菌种、结核分枝杆菌DNA多态性和北京基因型鉴定。结果80株菌株中非结核分枝杆菌占12.5%,70株结核分枝杆菌中北京家族占42.9%,非北京家族占57.1%。VNTR结果显示,菌株可分为5个基因群,其中 I群占15.7%,含 11个基因型,Ⅱ群占35.7%,含25个基因型,Ⅲ群占15.7%,含11个基因型,Ⅳ群占30.0%,含 21个基因型,Ⅴ群占2.9%,含2个基因型,未见成簇菌株。结论初步证实罗甸县结核分枝杆菌存在基因多态性,Ⅱ群、Ⅳ群为当地主要流行群,同时存在一定比例的非结核分枝杆菌感染。     

Virulence of Mycobacteria tuberculosis

The virulence of 43 Mycobacteria tuberculosis strains isolated from 21 patients with new cases of pulmonary tuberculosis and from 20 patients with chronic destructive pulmonary tuberculosis (CDPT) was studied in non-inbred albino mice. Twenty four (56%) and 19 (44%) M. tuberculosis strains belonged to the Beijing and individual genotypes, respectively. The virulence of isolates from the new cases of pulmonary tuberculosis was higher than that from CDPT patients. The M. tuberculosis strains studied showed no correlation between the virulence, viability, and affiliation to a certain genotype. However, there was a lower rate of decreased virulence in multidrug-resistant strains of the Beijing genotype than in the isolates of individual genotypes. This is likely to be a factor of the high transmissible capacity of M. tuberculosis strains of the Beijing family.     

Predominace of a novel Mycobacterium tuberculosis genotype in the Delhi region of India

Using IS 6110 -restriction fragment length polymorphism (RFLP) and spoligotyping, genetic variations of 83 Mycobacterium tuberculosis strains isolated from tuberculosis patients from two wards in a hospital in Delhi and a rural chest clinic near Delhi were analysed. The vast majority of the isolates (75%) were closely related and this novel genogroup was designated the 'Delhi type'. Both drug-sensitive and drug-resistant strains were found among strains of this genogroup. A minority of the strains harboured a single IS 6110 copy and only one strain belonged to the Beijing genotype, a genotype that is predominant in other parts of Asia. A comparison of the RFLP and spoligotype with existing data suggests that the predominance of Delhi genogroup is geographically limited to the Indian subcontinent and perhaps to specific regions in India. Despite the high prevalence of the M. tuberculosis strains of the Delhi type, the strains could easily be discriminated due to polymorphisms in the IS 6110 patterns. Future studies may disclose the genetic characteristics of strains belonging to the Delhi genotype, analogous to the recently observed virulence among the Beijing genogroup.     

DNA指纹技术检测耐多药结核分枝杆菌的应用研究

目的应用结核分枝杆菌DNA指纹技术,了解耐多药结核分枝杆菌的基因型状况并探讨耐多药结核病的分子流行病学。方法以结核分枝杆菌插入序列IS6110序列为模板,设计一对特异外向引物,建立一种结核分枝杆菌DNA指纹技术方法,对临床标本中分离的220株耐多药结核分枝杆菌进行DNA指纹基因分析。同时对实验数据进行聚类分析,进行耐多药结核病分子流行病学研究。结果根据结核分枝杆菌DNA指纹图谱分析,220株耐多药结核分枝杆菌均产生特征DNA指纹图谱,DNA指纹图谱变异很大。每一菌株DNA指纹的拷贝数介于3至18之间。其中大部分菌株,即205株（93.2%）包含613个拷贝,平均为11个带。这205株DNA指纹图谱中,162株为特异的,提示它们在流行病学上是独立的。有58株（26.4%）指纹图谱组成了15个簇,每簇28株,它们的IDNA指纹图谱相同,可能代表了近期的传播。尤其是在此DNA指纹图谱中,其中分子量为200 bp扩增带频率最大,在220株耐多药结核分枝杆菌均出现。结论结核杆菌DNA指纹技术检测可应用耐多药结核分枝杆菌DNA指纹图谱分型,并可用于耐多药结核病分子流行病学调查。