多位点数目可变串联重复序列分析在北京基因型结核分枝杆菌基因分型中的应用

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.     

间隔区寡核苷酸分型法用于99株结核分枝杆菌杭州临床分离株的基因分型研究

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。     

河南省结核分枝杆菌基因分型与耐药性分析

摘要:目的：研究河南省结核分枝杆菌基因型别与耐药性和地区分布关系。 方法：用间隔区寡核苷酸序列（Spoligotyping）基因分型技术对344株结核分枝杆菌进行分型。 结果：344株结核分枝杆菌分为4个基因群［Beijing和Beijing近似群(简称北京群)、T群、Manu2群、S和LAM3群］,共24个基因型,北京基因型有299株,占87.7%,但北京型与非北京型之间耐多药（MDR）株所占比例差异无统计学意义（χ²=1.46,P>0.05）;北京基因型在≤60岁感染者中分离率占83.6%;除个别地区外,不同地区间的分布无显著性差异。 结论：河南省结核分枝杆菌以北京基因型为主要流行型,与MDR和任意耐药无相关性,以≤60岁感染者居多,嵩县分离率略低。     

骨结核临床分离菌株基因分型的特点

目的对小样本量骨结核临床分离菌株基因型的特点进行初步研究,以了解引起骨结核与肺结核的病原菌在基因型方面有无差异,为探讨两者之间的关系提供相关依据。方法利用间隔区寡核苷酸分型法(Spoligotyping)和数目可变串联重复序列分析(VNTR)分型法对骨结核与肺结核的临床分离菌株进行分型,从而确定每株结核分枝杆菌的基因型。结果 Spoligotyping分型方法确定了在14株肺结核临床分离菌株中有9株为Beijing基因型,2株为T2基因型,另外3株分别为MANU2型、Haaslem3型以及未知型,VNTR可将14株肺结核临床分离菌株完全区分为14个基因型。而对于14株骨结核临床分离菌株来说,Spoligotyping和VNTR两种分型方法的结果均显示其具有完全相同的基因型。结论骨结核与肺结核临床分离菌株基因型差异巨大。特定型别(Spoligotyping:777777777777771,VNTR:013582202252)的结核分枝杆菌在骨结核流行菌株中占据主要地位,较之其他型别更易引起骨组织的病变。     

新疆维吾尔自治区与甘肃省部分地区结核分枝杆菌分子流行病学特征比较研究

目的 比较研究新疆维吾尔自治区与甘肃省部分地区结核分枝杆菌分子流行病学特征。 方法 2008年3-12月从新疆维吾尔自治区与甘肃省的3个县(区)收集痰涂片阳性的结核病患者的临床分离株。收集患者的性别、民族、病史、菌株耐药谱和菌株来源资料。药物敏感性实验采用比例法。运用实时荧光定量PCR(Realtime PCR)和结核分枝杆菌散在分布重复单位(MIRU-VNTR)基因分型方法鉴定北京基因型及亚型菌株并绘制系统发育树。 结果 在146份结核分枝杆菌分离株中,北京/W 系结核分枝杆菌84株,占57.5%。其中较现代的W菌/典型北京家族菌株66株,占78.6%。MIRU-VNTR基因分型方法将146株菌株分成140个基因型,其中包括134个独特型,6个成簇的基因型。 结论 在甘肃采样地区,北京/W 系的流行呈现明显优势,其中较为现代的W 菌/典型北京家族菌株的流行又呈较为明显的优势;而在新疆采样地区,以非北京/W 系菌株流行为主,两地结核分枝杆菌分子流行病学特征有明显区别。     

新疆结核分枝杆菌临床分离株Spoligotyping基因型的初步研究

目的研究新疆结核分枝杆菌Spoligotyping基因分型,初步了解其基因型多态性状况及主要流行株。方法在新疆维吾尔自治区胸科医院收集一个连续时间段的结核分枝杆菌临床分离菌株,采用比例法检测进行耐药性检测、间隔寡核苷酸分型(Spoligotyp ing)方法进行分型研究。基因聚类分析采用B ioNum erics 5.0数据库软件,统计学分析采用χ2检验,P0.05为差异有统计学意义。结果共收集到结核分枝杆菌临床分离菌株175株,其中对利福平、异烟肼、链霉素和乙胺丁醇全敏感115株,耐药60株(包括单耐药31株和耐多药29株)。经Spoligotyp ing分型,这些菌株可分为4个基因群49种基因型,最大的1个基因群为北京家族,占68.57%(120/175)。北京家族中,敏感菌株63.87%(76/119),耐药菌株占36.13%(43/119);非北京家族中,敏感菌株68.52%(37/54),耐药菌株占31.48%(17/54),北京家族与非北京家族的耐药率之间差异无统计学意义(P=0.551)。结论新疆结核分枝杆菌临床菌株存在明显的基因多态性,主要流行菌株为北京基因型。北京基因型与耐药性无明显相关性。     

内蒙古地区结核分枝杆菌串联重复序列基因多态性分析

目的 了解内蒙古地区结核分枝杆菌临床分离株的串联重复序列(variable number tandem repeat,VNTR)基因多态性和VNTR基因型构成,及不同VNTR位点在该地区结核分枝杆菌基因分型中的应用。 方法 从内蒙古自治区结核病防治研究所收集临床分离的结核分枝杆菌菌株及其病例背景资料,采用多位点串联重复序列分析(MLVA)对收集的菌株进行22个VNTR 位点分析,计算Hunter-Gaston 指数(HGDI),分析各个位点的分辨率, 同时用BioNumerics 软件对VNTR结果进行聚类分析。且统计学分析民族与主要基因型间的关系。 结果 372株菌共分为308个基因型,47簇,261个独特基因型,成簇率为17.20%。22个VNTR位点的基因多态性存在较大的差异,位点VNTR3820(HGDI 0.838)的分型分辨能力最高,MIRU23(HGDI 0.068)和MIRU27(HGDI 0.083)分型分辨能力较差。随着VNTR 位点的增加,分型的分辨能力也有所提高。Ⅰ群基因型菌株与民族易感性的分析表明,汉族与蒙古族间Ⅰ群基因型菌株分布差异无统计学意义(χ2=0.337, P=0.561>0.05)。 结论 内蒙古地区结核分枝杆菌临床分离株基因具有多态性,不同VNTR位点在内蒙古地区结核分枝杆菌中具有不同的分辨能力。且Ⅰ群基因型为该地区主要流行株,而Ⅰ群基因型菌株与民族易感性间无关联。     

结核分枝杆菌DNA指纹技术及其应用研究

目的　从分子流行病学角度探讨北京及其他地区结核分枝杆菌(结核菌)的分布特征。方法　构建以IS6110为基础的结核菌DNA指纹图谱,应用MINTS软件进行处理,并用χ2检验比较不同组别结核病人临床分离菌株成簇率的差别。结果　H37Rv、BCG两个标准菌株和62例结核病人临床分离菌株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1.00～0.65之间;分组统计结果显示,男性组与女性组成簇率之间差异有显著性（P<0.05）,其他各组之间差异无显著性（P>0.05）。结论　DNA指纹技术对结核菌在株水平的鉴定具有特异、灵敏等优点,可应用于结核流行病学研究。研究表明,北京及其他地区结核菌临床分离菌株多数遗传关系较近,且在基因水平上相关程度较强;结核菌在男性人群中的传播频率可能较女性更高。     

浙江省宁波地区耐多药结核分枝杆菌北京基因型的流行及与喹诺酮耐药关系的研究

目的 初步探讨北京基因型耐多药结核分枝杆菌在宁波地区的流行现况及与喹诺酮类药物耐药相关性,为本地区耐多药结核病的有效防控及分子流行病学研究提供科学依据。方法 应用RD105缺失基因检测法对宁波市疾病预防控制中心收集的106例耐多药结核分枝杆菌临床分离株（MDR-TB）进行北京基因型鉴定,并采用1%比例法对106例MDR-TB菌株进行3种喹诺酮类药物左氧氟沙星（LFX）、氧氟沙星（OFX）、莫西沙星（MXF）耐药性检测。结果 106株MDR-TB菌株中,北京基因型83株（78.3%,83/106）,非北京基因型23株（21.7%,23/106）。北京基因型耐喹诺酮类药物28株（33.7%,28/83）,非北京基因型耐喹诺酮类药物6株（26.1%,6/23）,差异无统计学意义（2=0.483,P0.05）。结论 宁波地区MDR-TB流行以北京基因型为主,MDR-TB对喹诺酮类药物的耐药与北京基因型无关。     

萍乡地区44株结核分枝杆菌药物敏感试验结果分析

目的探讨萍乡地区结核分枝杆菌的药物敏感性特点,更好地为临床结核病诊疗和流行病学调查提供科学依据。方法选用改良罗氏培养基,对痰标本结核分枝杆菌培养阳性菌株标本进行药物敏感试验。结果分离出结核分枝杆菌44株,其中耐药株34株、敏感株10株,耐药率77.3%。结论本地区结核耐药菌已普遍存在并已出现耐多药菌株,应重视结核分枝杆菌培养并根据药物敏感试验结果对结核病进行合理用药治疗。     

High prevalence of KatG Ser315Thr substitution among isoniazid-resistant Mycobacterium tuberculosis clinical isolates from northwestern Russia, 1996 to 2001

A total of 204 isoniazid (INH)-resistant strains of Mycobacterium tuberculosis isolated from different patients in the northwestern region of Russia from 1996 to 2001 were screened by a PCR-restriction fragment length polymorphism (RFLP) assay. This assay uses HapII cleavage of an amplified fragment of the katG gene to detect the transversion 315AGC-->ACC (Ser-->Thr), which is associated with INH resistance. This analysis revealed a 93.6% prevalence of the katG S315T mutation in strains from patients with both newly and previously diagnosed cases of tuberculosis (TB). This mutation was not found in any of 57 INH-susceptible isolates included in the study. The specificity of the assay was 100%; all isolates that contained the S315T mutation were classified as resistant by a culture-based susceptibility testing method. The Beijing genotype, defined by IS6110-RFLP analysis and the spacer oligonucleotide typing (spoligotyping) method, was found in 60.3% of the INH-resistant strains studied. The katG S315T shift was more prevalent among Beijing genotype strains than among non-Beijing genotype strains: 97.8 versus 84.6%, respectively, for all isolates, including those from patients with new and previously diagnosed cases, isolated from 1999 to 2001 and 100.0 versus 86.5%, respectively, for isolates from patients with new cases isolated from 1996 to 2001. The design of this PCR-RFLP assay allows the rapid and unambiguous identification of the katG 315ACC mutant allele. The simplicity of the assay permits its implementation into routine practice in clinical microbiology laboratories in regions with a high incidence of TB where this mutation is predominant, including northwestern Russia.     

中国结核分枝杆菌寡核苷酸基因分型及其耐药性分析

目的 选用间隔区寡核苷酸分型方法对有中国代表性的菌株进行基因分型,研究不同基因型在中国的流行情况,并分析基因型与耐药表型的关系.方法 中国疾病预防控制中心于2007-2008年,按照流行病学抽样的原则从中国31个省收集涂片阳性的结核病患者的临床分离株4 017株,采用比例法进行药物敏感性实验,选用寡核苷酸分型方法对菌株进行基因分型.基因型检出率差异比较采用x2检验.结果 在4017株结核分枝杆菌临床分离株中,北京基因型菌株包括2 500株(62.2％).北方地区来源菌株北京基因型检出率达76.5％(1 913株),高于南方地区检出率(53.2％,1 330株),差异有统计学意义(x2=219.69,P＜0.05).南方地区T1型检出率达13.3％(332株),高于北方地区检出率(4.3％,108株),差异有统计学意义(x2=88.07,P＜0.05).北京基因型在耐利福平(21.7％)、耐氧氟沙星(4.9％)和MDR( 11.3％)的比例都高于非北京基因型的相应菌株比例18.4％、2.4％和7.4％,差异有统计学意义(x2值分别为22.10、14.42和14.83,P均＜0.05).结论 北京基因型目前仍然是中国主要流行基因型,并且比例呈现出地域差别,北方地区显著高于南方地区.北京基因型菌株与耐利福平、耐氧氟沙星和MDR有关.     

Comparison between spoligotyping and IS6110 restriction fragment length polymorphisms in molecular genotyping analysis of Mycobacterium tuberculosis strains

Spoligotyping was compared with RFLP fingerprinting analysis in the identification of Mycobacterium tuberculosis strains. Spoligotyping sensitivity was 97.6% with a specificity of 47%. The global probability for two strains clustered with spoligotyping to be clustered also with RFLP analysis was 33%; the probability for two strains clustered with RFLP analysis to be clustered also with spoligotyping analysis was 95%. However, comparing the two methods in five outbreak episodes, full concordance was evidenced between spoligotyping and RFLP. Moreover, we evaluated the presence of our 17 largest spoligotyping clusters in spoligotyping databases from Caribbean countries, London and Cuba. Only five out of 17 patterns were present in all the cohorts. The conditional probability comparing spoligotyping and RFLP methods related to these patterns resulted in very low concordance (range from 2 to 38%). In conclusion, we confirm that spoligotyping when used alone overestimates the number of recent transmission and does not represent a suitable method for wide clinical practice application. However, it allows to get a first good picture of strain identity in a new setting and in more localized or confined settings, the probability of reaching the same result compared to RFLP was 100% confirming the usefulness of spoligotyping in the management of epidemic events, especially in hospitals, prisons and close communities.     

Evolution of drug resistance in different sublineages of Mycobacterium tuberculosis Beijing genotype

We compared the population structure and drug resistance patterns of the Mycobacterium tuberculosis strains currently circulating in the Beijing area of China. One hundred thirteen of 123 strains belonged to the Beijing family genotypes defined by spoligotyping. The Beijing genotype strains were further subdivided into old and modern sublineages on the basis of NTF locus analysis. A stronger association with resistance to the more recently introduced antituberculosis drugs has been observed for old versus modern strains of the Beijing genotype, suggesting that its different sublineages may differ in their mechanisms of adaptation to drug selective pressure.     

Comparison between molecular epidemiology, geographical regions and drug resistance in Mycobacterium tuberculosis strains isolated from Iranian and Afghan patients

The aim of this study was to determine the prevalence of drug-resistant tuberculosis (TB) and its associated risk factors. The susceptibilities of Mycobacterium tuberculosis isolates were tested against four first-line antituberculous drugs and were typed by spoligotyping. Spoligotyping of M. tuberculosis strains resulted in 95 different patterns that were divided into three evolutionary groups (1-3). Eighty-six (90%) of the isolates had unique patterns that were reported for the first time. Interestingly, 9.4% of the strains belonged to the Beijing family. Multidrug resistance (MDR) was seen in group 1 of the evolutionary scenario. All M. tuberculosis isolates belonging to the Beijing family were associated with a resistance pattern. MDR was much higher in bacteria isolated from Afghan TB patients residing in Iran.     

IS6110: conservation of sequence in the Mycobacterium tuberculosis complex and its utilization in DNA fingerprinting.

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.