两株结核分枝杆菌北京基因型菌株全基因组测序和初步分析

发布时间：2018-03-25 10:23

本文选题：结核分枝杆菌　切入点：北京基因型　出处：《中国疾病预防控制中心》2011年硕士论文

【摘要】：目的对两株结核分枝杆菌北京基因型临床分离菌株(CCDC5079和CCDC5180)进行全基因组测序,通过与其它8株已经完成全基因组测序的结核分枝杆菌复合群(Mycobacterium tuberculosis complex, MTBC)菌株数据进行比对分析,了解结核分枝杆菌北京基因型菌株的基因组分子特征,并结合菌株的药物敏感性信息,分析可能与耐药相关的基因组差异,为进一步研究提供基础。方法收集培养临床分离的结核分枝杆菌菌株,进行药物敏感性检测和分子分型鉴定,选取两株典型的北京基因型菌株,包括对三种一线抗结核药物(异烟肼、利福平和链霉素)全敏感和全耐药菌株各1株；在改良罗氏培养基上传代培养,收集菌体,用CTAB法提取全基因组DNA；用Roche454GS FLX测序仪进行全基因组高通量测序。测序结果用随测序仪提供的拼接软件进行初始序列的拼装,得到测序重叠群(contigs),与参考序列进行blast比对后,确定其位置关系和测序未覆盖区(gaps)的大小。测序未覆盖区和测序低值区,经特异性PCR扩增后,用ABI3730测序仪进行测序。基因组全长序列用Phred、Phrap和Consed软件包进行拼装和碱基修正,最终得到CCDC5079和CCDC5180两株的全基因组序列数据,与从NCBI genome网站下载的结核分枝杆菌复合群菌株的全基因组数据,在基因组水平进行比对、分析。结果经临床标本的分离培养、菌种鉴定、药物敏感性检测和分子分型等,确认两株典型结核分枝杆菌北京基因型临床分离菌株CCDC5079和CCDC5180,其中,CCDC5079对所有三种抗结核一线药物均敏感,CCDC5180为全耐药。测序得到CCDC5079和CCDC5180两株的全基因组大小分别为4414324bp和4414344bp,其基因组测序数据的准确度分别为99.9996%和99.9988%,预测的蛋白质编码序列(predicted protein coding sequences, CDSs)分别为4158个和4161个,CDSs平均大小分别为971bp和970bp。通过与已完成全基因组测序的MTBC菌株的全基因组数据的比对分析发现,在全基因组水平,存在结核分枝杆菌北京基因型特异性的609个单核苷酸多态性(Single-nucleotide polymorphism, SNP)位点和84个插入和缺失序列(Insertion and Deletion, Indel)位点;可能与结核分枝杆菌药相关的156个SNP位点,以及其它分子特征。结论成功完成了两株结核分枝杆菌北京基因型菌株的全基因组测序工作,为进一步研究结核分枝杆菌北京基因型菌株的基因结构特征和其分子进化提供了全基因组序列数据。对全基因组数据的初步分析发现,结核分枝杆菌北京基因型菌株基因组序列具有某些特异性的SNP、 Indel及其它分子生物学特征,这些分子生物学特征将为结核分枝杆菌北京基因型的快速鉴定、菌株耐药性检测及其耐药和致病机制研究等多个方面提供良好基础。并将促进对结核分枝杆菌北京基因型及结核分枝杆菌生物学本质的认识,对于最终实现控制结核病的目标具有重要意义。
[Abstract]:Purpose. Two strains of Mycobacterium tuberculosis, CCDC5079 and CCDC5180, were sequenced. The data of Mycobacterium tuberculosis complex (MTBCc) strains of Mycobacterium tuberculosis complex, which had completed the whole genome sequencing, were compared and analyzed. To understand the genomic molecular characteristics of Mycobacterium tuberculosis Beijing genotypes and to analyze the genomic differences related to drug resistance in combination with the drug sensitivity information of the strains, which provides a basis for further research. Method. Mycobacterium tuberculosis strains isolated from clinical culture were collected for drug sensitivity test and molecular typing identification. Two typical strains of Beijing genotype were selected, including three first-line anti-tuberculosis drugs (isoniazid, isoniazid, isoniazid). Rifampicin and streptomycin), one strain of allsensitive and one strain of total drug resistance were cultured in the modified Roche medium, and the bacteria were collected. The whole genome DNA was extracted by CTAB method, and the whole genome high-throughput sequencing was carried out by Roche454GS FLX sequencer. The sequence result was assembled with the splicing software provided by the sequencer, and the overlapping group contigsone was obtained. After comparing with the reference sequence, the sequence was compared with the blast sequence. The location relationship and the size of the uncovered region were determined. The uncovered region and the low value region were sequenced. After the specific PCR amplification, the whole genome sequence was sequenced by ABI3730 sequencer. The whole genome sequence was assembled and modified by PhredPrap and Consed software package. Finally, the whole genome sequence data of CCDC5079 and CCDC5180 strains were obtained and compared with the whole genome data of Mycobacterium tuberculosis complex strains downloaded from NCBI genome website. Results. After isolation and culture of clinical specimens, identification of bacteria, detection of drug sensitivity and molecular typing, etc. Two typical strains of Mycobacterium tuberculosis, CCDC5079 and CCDC5180, were identified, and CCDC5079 was sensitive to all three anti-TB first-line drugs. The whole genome size of CCDC5079 and CCDC5180 were 4414324bp, respectively. The accuracy of genome sequencing data was 99.9996% and 99.9988%, respectively, and the predicted protein coding sequence protein sequences (CDSs) were 4158 and 4161 971bp and 970 BP, respectively. Analysis of the whole genome data found that, At the whole genome level, there were 609 single-nucleotide polymorphisms (SNPs) and 84 insertion and deletion and deletion sites in Mycobacterium tuberculosis, and 156 SNP sites that might be associated with Mycobacterium tuberculosis drugs. And other molecular characteristics. Conclusion. The complete genome sequencing of two strains of Mycobacterium tuberculosis Beijing genotype was successfully completed. In order to further study the gene structure characteristics and molecular evolution of Mycobacterium tuberculosis Beijing genotype strain, the whole genome sequence data were provided. The genomic sequence of Mycobacterium tuberculosis Beijing genotype strain has some specific SNPs, Indel and other molecular biological characteristics, which will be the rapid identification of Mycobacterium tuberculosis Beijing genotype. The detection of drug resistance and the study of drug resistance and pathogenicity of the strains provide a good basis for the understanding of the Beijing genotype of Mycobacterium tuberculosis and the biological nature of Mycobacterium tuberculosis. It is of great significance to achieve the goal of tuberculosis control.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378.911;R3416

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