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  本文選題：結(jié)核分枝桿菌　切入點：北京基因型　出處：《中國疾病預(yù)防控制中心》2011年碩士論文

【摘要】：目的 對兩株結(jié)核分枝桿菌北京基因型臨床分離菌株(CCDC5079和CCDC5180)進(jìn)行全基因組測序,通過與其它8株已經(jīng)完成全基因組測序的結(jié)核分枝桿菌復(fù)合群(Mycobacterium tuberculosis complex, MTBC)菌株數(shù)據(jù)進(jìn)行比對分析,了解結(jié)核分枝桿菌北京基因型菌株的基因組分子特征,并結(jié)合菌株的藥物敏感性信息,分析可能與耐藥相關(guān)的基因組差異,為進(jìn)一步研究提供基礎(chǔ)。 方法 收集培養(yǎng)臨床分離的結(jié)核分枝桿菌菌株,進(jìn)行藥物敏感性檢測和分子分型鑒定,選取兩株典型的北京基因型菌株,包括對三種一線抗結(jié)核藥物(異煙肼、利福平和鏈霉素)全敏感和全耐藥菌株各1株；在改良羅氏培養(yǎng)基上傳代培養(yǎng),收集菌體,用CTAB法提取全基因組DNA；用Roche454GS FLX測序儀進(jìn)行全基因組高通量測序。測序結(jié)果用隨測序儀提供的拼接軟件進(jìn)行初始序列的拼裝,得到測序重疊群(contigs),與參考序列進(jìn)行blast比對后,確定其位置關(guān)系和測序未覆蓋區(qū)(gaps)的大小。測序未覆蓋區(qū)和測序低值區(qū),經(jīng)特異性PCR擴(kuò)增后,用ABI3730測序儀進(jìn)行測序�；蚪M全長序列用Phred、Phrap和Consed軟件包進(jìn)行拼裝和堿基修正,最終得到CCDC5079和CCDC5180兩株的全基因組序列數(shù)據(jù),與從NCBI genome網(wǎng)站下載的結(jié)核分枝桿菌復(fù)合群菌株的全基因組數(shù)據(jù),在基因組水平進(jìn)行比對、分析。 結(jié)果 經(jīng)臨床標(biāo)本的分離培養(yǎng)、菌種鑒定、藥物敏感性檢測和分子分型等,確認(rèn)兩株典型結(jié)核分枝桿菌北京基因型臨床分離菌株CCDC5079和CCDC5180,其中,CCDC5079對所有三種抗結(jié)核一線藥物均敏感,CCDC5180為全耐藥。測序得到CCDC5079和CCDC5180兩株的全基因組大小分別為4414324bp和4414344bp,其基因組測序數(shù)據(jù)的準(zhǔn)確度分別為99.9996%和99.9988%,預(yù)測的蛋白質(zhì)編碼序列(predicted protein coding sequences, CDSs)分別為4158個和4161個,CDSs平均大小分別為971bp和970bp。通過與已完成全基因組測序的MTBC菌株的全基因組數(shù)據(jù)的比對分析發(fā)現(xiàn),在全基因組水平,存在結(jié)核分枝桿菌北京基因型特異性的609個單核苷酸多態(tài)性(Single-nucleotide polymorphism, SNP)位點和84個插入和缺失序列(Insertion and Deletion, Indel)位點;可能與結(jié)核分枝桿菌藥相關(guān)的156個SNP位點,以及其它分子特征。 結(jié)論 成功完成了兩株結(jié)核分枝桿菌北京基因型菌株的全基因組測序工作,為進(jìn)一步研究結(jié)核分枝桿菌北京基因型菌株的基因結(jié)構(gòu)特征和其分子進(jìn)化提供了全基因組序列數(shù)據(jù)。對全基因組數(shù)據(jù)的初步分析發(fā)現(xiàn),結(jié)核分枝桿菌北京基因型菌株基因組序列具有某些特異性的SNP、 Indel及其它分子生物學(xué)特征,這些分子生物學(xué)特征將為結(jié)核分枝桿菌北京基因型的快速鑒定、菌株耐藥性檢測及其耐藥和致病機(jī)制研究等多個方面提供良好基礎(chǔ)。并將促進(jìn)對結(jié)核分枝桿菌北京基因型及結(jié)核分枝桿菌生物學(xué)本質(zhì)的認(rèn)識,對于最終實現(xiàn)控制結(jié)核病的目標(biāo)具有重要意義。
[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.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R378.911;R3416

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