广东省结核病流行菌株研究

目的：从分子流行病学的角度探讨广东省结核分支杆菌菌株流行的分布。方法：构建广东省74株临床分离的以RFLP为基础的结核分支杆菌的IS6110 DNA指纹图谱技术，确诊结核分支杆菌菌株的流行分布。结果：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”结核分支杆菌菌株。结论：广东省结核菌株流行主要存在着遗传关系接近，在基因水平上相关程度较高的“北京家族”结核分支杆菌菌株，且该家族菌株正以一定的比例在我省流行。     

Determination of drug susceptibility and DNA fingerprint patterns of clinical isolates of Mycobacterium tuberculosis from Kampala, Uganda

This study was undertaken to determine the rate of initial drug resistance and transmission patterns of Mycobacterium tuberculosis (TB) in Kampala, Uganda. Using a radiometric BACTEC 460 TB system, 215 M. tuberculosis isolates from previously untreated patients (aged 17-48 years, mean age = 28 years; 56% males and 69% HIV-seropositive) were analyzed for susceptibility to isoniazid, rifampin, streptomycin, and ethambutol. Isolates from 73 patients were examined for strain diversity or relatedness using the insertional sequence 6110 (IS6110) DNA fingerprinting technique. The study revealed the following drug resistance rates: isoniazid, 7.9%; streptomycin, 6.1%; rifampin, 1.4%; and ethambutol, 0.9%. Resistance to at least one of the first line drugs tested were developed by 12% of the strains, while 4.7% showed multiple resistance. However, no significant differences in resistance rates were found between patients with and without HIV infection. Using the number and size of DNA fragments containing IS6110, only three clusters of isolates with identical patterns were found out of the 73 isolates tested (8.2%). Each cluster contained two isolates, and three isolates had less than 7 copies of IS6110. These findings suggest that initial drug resistance to anti-tuberculosis agents in this region is low and similar to other countries in sub-Saharan Africa and that multiple strains of M. tuberculosis have been transmitted within the community.     

Evaluation of four candidate VNTR Loci for genotyping 225 Chinese clinical Mycobacterium tuberculosis complex strains

Objective To evaluate four candidate variable number tandem repeat (VNTR) loci for genotyping Mycobacterium tuberculosis complex strains.Methods Genomic sequences for two M.tuberculosis strains (CCDC5079 and CCDC5180) were generated,and using published sequence data,four candidate VNTR loci were identified.The VNTRs were used to genotype 225 Chinese clinical M.tuberculosis complex strains.The discriminatory power of the VNTRs was evaluated using BioNumerics 5.0 software. Results The Hunter-Gaston Index (HGI) for BJ1,BJ2,BJ3,and BJ4 loci was 0.634,0.917,0.697,and 0.910,respectively.Combining all four loci gave an HGI value of 0.995,thus confirming that the genotyping had good discriminatory power.The HGI values for BJ1,BJ2,B J3,and BJ4,obtained from Beijing family strain genotyping,were 0.447,0.878,0.315,and 0.850,respectively.Combining all four loci produced an HGI value of 0.988 for genotyping the Beijing family strains.We observed unique patterns for M.bovis and M.africanum strains from the four loci.Conclusion We have shown that the four VNTR loci can be successfully used for genotyping M.tuberculosis complex strains.Notably,these new loci may provide additional information about Chinese M.tuberculosis isolates than that currently afforded by established VNTR loci typing.     

结核分枝杆菌IS6110和IS1081微滴数字PCR检测体系的建立和应用

目的 建立结核分枝杆菌IS6110和IS1081微滴数字PCR(droplet digital PCR,ddPCR)检测体系,并用于不同类型临床样本的检测.方法 常规提取13株结核分枝杆菌和14株非结核分枝杆菌临床分离株DNA,9例结核分枝杆菌阳性肺结核患者和4例其他肺部疾病患者痰DNA,12例结核分枝杆菌阳性肺结核患者和7例健康者血浆DNA.设计合成IS6110和IS1081扩增引物和检测探针,建立ddPCR检测体系.应用该体系检测分枝杆菌、痰和血浆3种DNA样本中靶标拷贝数,进行统计学分析.结果 建立了能同时检测IS6110和IS1081的ddPCR体系,该体系检测2个靶标的线性范围分别为0.5～8 733和0.2～2 893拷贝/μl反应体系.该体系具有较好的重复性(r＞0.95),以非结核分枝杆菌为对照检测结核分枝杆菌的灵敏度和特异度均为100％.在痰和血浆DNA样本检测中,2个靶标拷贝数在肺结核组均显著高于对照组.结论 结核分枝杆菌特异性核酸的ddPCR体系,可用于肺结核患者临床分离株、痰和血浆样本的微量核酸检测,对提高结核病病原学诊断能力有重要意义.     

应用PCR方法扩增IS6110保守片段快速鉴定结核分支杆菌

目的：了解PCR方法快速鉴定结核分支杆菌的可靠性，探索快速诊断结核病的实验方法。方法：应用PCR方法扩增149株结核分支杆菌临床分离株的IS6110保守片段，并与结核分支杆菌H37Rv标准株进行比较。结果：149株临床分离株中，140株可以扩增出与结核分支杆菌H37Rv标准株相同的123bp DNA片段(敏感性达93．9％)，而阴性对照均不能扩增出该片段。结论：应用PCR方法扩增IS6110保守片段来鉴定结核分支杆菌，结果准确可靠，且具有方便和快速等优点，有较大的临床应用价值。     

结核分枝杆菌基因分型及其耐药性分析

目的探讨四川地区结核患者结核分枝杆菌基因分型及其与耐药性关系。方法采用PCR和琼脂糖凝胶电泳技术,对124株结核分枝杆菌的6个可变重复位点进行检测,并应用Gel-Proanalyzer3.0软件和BioNumerics（Version3.0）软件对检测结果进行基因型分析,同时分析其基因型与耐药之间的关系。结果124株结核分枝杆菌共分为37个不同的VNTR类型,成簇基因型占74.19%（92/124）,单一基因型占25.81%（32/124）。耐药菌株在成簇类型和单一类型的分布上存在统计学差异。结论四川地区结核患者的结核分枝杆菌具有明显的基因多态性,成簇类型是主要流行菌株,应加强流行结核菌株的监控。     

海南地区484株结核分枝杆菌菌株基因型特征分析

目的 了解海南地区结核分枝杆菌不同基因型流行特征,探讨基因分型用于评价本地区结核病控制中的应用价值。方法 本研究选取海南医学院第二附属医院自2013-2016年所有培养阳性结核分枝杆菌484株,采用SNP和Gao等推荐的12位点可变数目串联重复序列（variable number tandem repeat, VNTR）进行基因分型。结果 2013-2016年期间,484株结核分枝杆菌菌株中,北京基因型菌株279株,占57.6%,非北京基因型菌株205株,占42.4%,北京基因型菌株中包括91株(32.6%)古代北京基因型和188株(67.4%)现代北京基因型。北京基因型所占比例在四年间无显著变化,从2013年的59.6%到2016年的57.6%。采用3个高变VNTR位点基因分型后,成簇菌株数为0。结论 海南地区结核分枝杆菌存在明显的VNTR基因多态性,主要流行株仍以北京基因型为主,北京基因型所占比例在四年期间无显著性变化,且菌株成簇率较低,提示近期传播率低。     

Molecular epidemiology of Mycobacterium tuberculosis in Gansu province of China

Background  Mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) and Beijing family typing based on detecting the deletion of RD105 sequence are two common genotyping methods used to study the molecular epidemiologic characteristics of Mycobacterium (M.) tuberculosis. We collected 218 strains of M. tuberculosis between 2004 and 2006 in the Linxia Hui Autonomous Prefecture of Gansu province in Northwest China.

Methods  MIRU-VNTR analysis and Beijing family typing based on detecting the deletion of RD105 sequence were used to type the 218 strains, and their typing power was evaluated to look for practical and efficient genotyping methods suitable for the region.

Results  The MIRU typing yielded 115 distinct genotypes, including 98 unique isolates and 17 different clusters containing 120 isolates (55.05%); the cluster rate was 47.25%. By detecting the deletion of RD105 sequence, 188 of 218 (86.23%) isolates belonged to Beijing family. Combination of Beijing family typing and MIRU typing yielded 118 distinct patterns, including 101 unique isolates and 17 clusters containing 117 isolates (54.13%). The largest cluster contained 58 strains with MIRU genotype of 223325173533 which contained 50 strains belonging to Beijing family and 8 strains belonging to non-Beijing family.

Conclusions  The Beijing family strains occupied a large proportion and the Beijing family MIRU genotype 223325173533 is a dominant strain in Linxia of Gansu. Combining detecting the deletion of RD105 and MIRU typing together provides a simple, fast, and effective method which is low in cost and might be practical and suitable for M. tuberculosis genotyping in China.

     

某矿区尘肺结核与单纯肺结核分离株的基因型比较

目的比较某矿区尘肺结核与单纯肺结核分离株的基因型差异。方法42株结核分枝杆菌分离自某矿区的尘肺结核和肺结核患者,其中尘肺结核16株,单纯肺结核26株。采用MIRu技术和BioNumerics3．0聚类分析软件进行基因分型和成簇性分析。结果12个MIRU位点多态性分型结果表明,尘肺结核与单纯肺结核分离株在第26、40MIRU位点的基因多态性较高,聚类分析结果证实两者的分离株具有不同的成簇性特征。结论提示煤工尘肺结核患者中流行的结核杆菌可能具有独立性。     

吉林省110株结核分枝杆菌临床分离株MLVA基因分型

目的：利用多位点可变数目串联重复序列(MLVA)分型技术,了解近期吉林省结核分枝杆菌临床分离株基因型分布情况。方法：收集分离自吉林省2011-2012年不同市县来源患者标本的结核分枝杆菌分离株,提取基因组DNA,对12个数目可变串联重复序列（VNTR）位点进行扩增和产物电泳分析,使用Totalab软件对电泳条带进行数据化分析,使用BioNumerics（6.0）软件获得聚类分析结果。结果：对110株结核分枝杆菌临床分离株的12个VNTR位点进行检测,结果显示这些菌株有明显的多态性。12个VNTR位点中Hunter-Gaston指数能达到0.5以上的VNTR位点有9个,位点分辨能力最高的是MIRU26,最低的是MIRU20。基于12个位点的聚类分析,110株分离株可分为6个基因型群,其中分布最多是5群,占34％;其次是1群,占15％。分布最多的5群主要流行于吉林省通化市和松原市等地区。结论：吉林省流行的菌株存在着明显的遗传多态性;不同市县分布的主要基因型群存在差异,表明吉林省内不同地区结核分枝杆菌基因型的流行趋势不同。