基于可变数目串联重复序列分型方法的结核分子流行病学研究

发布时间：2018-07-03 01:16

本文选题：结核分枝杆菌 + 基因分型　；参考：《天津医科大学》2007年硕士论文

【摘要】： 目的：初步探讨15个可变数目串联重复(Variable Number Tandem Repeat，VNTR)位点在天津地区结核分枝杆菌(Mycobacterium tuberculosis，MTB)中的多态性、天津地区的主要流行菌株以及VNTR分型方法在结核分子流行病学中的主要应用。方法：选取天津市结核病控制中心参比实验室菌种库中2005年6月至2006年4月的100株MTB，收集这100例确诊肺结核患者的一般资料和实验室资料。将菌株复苏传代，提取其基因组DNA。采用PCR方法扩增结核菌基因组中的15个VNTR位点，包括由12个结核分枝杆菌散在重复单元(Mycobacterial Interspersed Repetitive Unit，MIRU)和5个精确串联重复序列(Exact Tandem Repeats，ETR)(其中的两个与MIRU位点一致)。扩增的产物经琼脂糖凝胶电泳后，应用Bio-Rad凝胶成像系统观察PCR产物并通过Quantity One4.4分析软件确定PCR产物的片段长度并与标准菌株H_(37)Rv比较计算得到每个菌株不同串联重复位点的拷贝数，将部分菌株的VNTR位点PCR扩增产物经纯化后直接测序；评价每个VNTR位点的多态性以及MIRU位点和ETR位点对天津地区结核菌株的综合分辨能力。应用PAUP4.0软件绘制系统树图，揭示菌株之间的相互关系。根据VNTR基因分型结果，设计调查表，收集成簇菌株的患者资料，用以追踪同一簇患者间的相互传播途径。应用SPSS11.5进行卡方检验和单因素Logistic回归分析。结果： 1、基本资料描述：本次研究中的100株MTB分别来自100名肺结核患者，患者的男女比例为1.9：1，，年龄的中位数为36岁(15-82岁)，以20-29岁患者比例最高。市内六区患者占患者总数的81％，初治患者占81％，有卡介苗接种史的患者占68％。 2、本次研究中58株MTB的5个ETR-VNTR位点拷贝数编码均为42435，另有两株编码分别为42436和42433，均属于具有北京家族VNTR分型特点的菌株，共占实验菌株总数的60％。 3、5个ETR-VNTR位点仅将菌株分为23个基因型，独立株17株，6个簇，最大簇包含58株菌；12个MIRU-VNTR位点将菌株分为45个基因型，独立株36株，9个簇，最大簇包含34株菌；MIRU和ETR-VNTR相结合后将菌株可被分为52个基因型，独立株41株，11个簇，最大簇包含29株菌。成簇组与非成簇组患者在性别，年龄，住址，治疗史，卡介苗接种史等方面的差异均无统计学意义(P＞0.05)。 4、VNTR位点等位基因多样性(h)分析结果显示：MIRU-26和MIRU-31位点多态性较好h分别为0.54和0.45；其次为MIRU-10，40和ETR-A，0.3＜h＜0.4；MIRU-39，27，0.2＜h＜0.3；MIRU-4，16，23和ETR-C，0.1＜h＜0.2；MIRU-2，20，24和ETR-B的多样性最低h＜0.1。不同VNTR位点组合对结核分枝杆菌的分辨能力比较结果显示：单独使用5个ETR位点、12个MIRU位点和两者结合后分辨能力HGDI分别为0.6503，0.8622和0.9093。 5、PAUP分析表明，100株MTB可被分为三组(Ⅰ，Ⅱ，Ⅲ)，其中89株菌属于第Ⅲ组，属于Ⅰ，Ⅱ两组的菌株分别为6株和5株。 6、对成簇组的结核病患者资料进一步调查发现，其中2名患者为相继发病的某中学同班同学，可以确定这两名患者处于同一条传播链上。结论：15个VNTR位点中MIRU-10，26，31，40和ETA五个位点在天津地区流行的MTB中多态性较好，其余位点对菌株的分辨能力欠佳。MIRU和ETR-VNTR位点还不足以达到理想的分型效果，还应再引入其他多态性位点。天津地区可能是北京家族菌株的高流行区，应加强对该类菌株的预防和控制。VNTR分型方法可广泛用于结核病的分子流行病学研究。
[Abstract]:Objective: To investigate the polymorphism of the 15 Variable Number Tandem Repeat (VNTR) loci in the Mycobacterium tuberculosis (MTB) in Tianjin area, and the main epidemic strains in Tianjin and the main application of the VNTR typing method in the epidemiology of tuberculosis.
Methods: 100 strains of MTB from June 2005 to April 2006 in the reference laboratory of the Tianjin tuberculosis control center were selected to collect the general data and laboratory data of the 100 cases of pulmonary tuberculosis. The strains were resuscitation, and the genomic DNA. was extracted by PCR method to amplify 15 VNTR loci in the genome of tuberculosis bacteria, including 12 Mycobacterium tuberculosis scattered in the repeating unit (Mycobacterial Interspersed Repetitive Unit, MIRU) and 5 precise series repeats (Exact Tandem Repeats, ETR). (two of them are in agreement with the MIRU loci). The amplified products were analyzed by the agarose gel electrophoresis, and the PCR products were observed and analyzed by the Bio-Rad gel imaging system. The software determined the length of the fragment of the PCR product and compared with the standard strain H_ (37) Rv to calculate the copy number of the different tandem reset points of each strain. The PCR amplification products of the VNTR loci of some strains were directly sequenced, and the polymorphism of each VNTR site and the synthesis of the MIRU and ETR loci in the synthesis of the tuberculosis strains in Tianjin region were evaluated. PAUP4.0 software was used to map the system tree map to reveal the relationship between the strains. According to the results of the VNTR genotyping, the questionnaire was designed and the patient data of the cluster strains were collected to track the communication pathways of the same cluster of patients. SPSS11.5 was used for chi square test and single factor Logistic regression analysis.
Result:
1, basic data description: 100 MTB in this study were from 100 tuberculosis patients, the proportion of men and women was 1.9:1, the median age was 36 years (15-82 years old), the highest proportion was 20-29 years old. The six patients in the city accounted for 81% of the total, the initial treatment was 81%, and the patients with the history of BCG vaccination accounted for 68%..
2, in this study, the number of 5 ETR-VNTR loci copies of 58 MTB was 42435, and the other two strains were 42436 and 42433, all of which belonged to the VNTR typing of the Beijing family, which accounted for 60%. of the total number of experimental strains.
3,5 ETR-VNTR loci only divide the strain into 23 genotypes, 17 independent strains, 6 clusters and 58 strains. 12 MIRU-VNTR sites divide the strains into 45 genotypes, 36 isolated strains, 9 clusters, and 34 strains, and the strain can be divided into 52 genotypes, 41 strains of independent strains, 11 clusters, and the largest clusters after MIRU and ETR-VNTR. There were 29 strains. There was no significant difference in the sex, age, address, treatment history and BCG vaccination history between the cluster group and the non clustered group (P > 0.05).
4, VNTR allele diversity (H) analysis showed that the better h of MIRU-26 and MIRU-31 loci were 0.54 and 0.45, followed by MIRU-10,40 and ETR-A, 0.3 < h < 0.4, MIRU-39,27,0.2 < 0.3, MIRU-4,16,23 and ETR-C, 0.1 < 0.2. The results of the resolution of Mycobacterium tuberculosis showed that 5 ETR loci alone, 12 MIRU loci and the binding post resolution HGDI were 0.6503,0.8622 and 0.9093., respectively.
5, PAUP analysis showed that 100 strains of MTB could be divided into three groups (I, II, III), of which 89 strains belonged to group III, belonging to group I and II two strains were 6 strains and 5 strains respectively.
6, a further investigation of the data of tuberculosis patients in the cluster group found that 2 of the patients were classmate of a secondary school who had been attacked successively, and the two patients could be determined to be in the same transmission chain.
Conclusion: the five loci in the 15 VNTR loci are better polymorphic in the popular MTB in Tianjin area, and the other loci are not good enough to distinguish the strains. The.MIRU and ETR-VNTR loci are not enough to achieve the ideal typing effect, and the other polymorphic loci should be reintroduced. The Tianjin region may be the high prevalence of the Beijing family strain. The prevention and control of this strain should be strengthened. The.VNTR typing method can be widely used in molecular epidemiological studies of tuberculosis.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R181.3

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