本文選題：結(jié)核分枝桿菌 + EAI基因型�。� 參考：《中國疾病預(yù)防控制中心》2012年碩士論文

【摘要】：目的對中國結(jié)核分枝桿菌EAI和CAS基因型臨床分離菌株(CCDC06029和CCDC06153)全基因組進(jìn)行測序,并與其它已經(jīng)完成全基因組測序的結(jié)核分枝桿菌復(fù)合群菌株的數(shù)據(jù)進(jìn)行比對分析,了解EAI和CAS基因型菌株的全基因組分子特征,為進(jìn)一步深入研究,如微進(jìn)化、毒力學(xué)等,提供基礎(chǔ)。方法選取經(jīng)細(xì)菌學(xué)、藥物敏感性和分子分型鑒定的中國結(jié)核分枝桿菌EAI和CAS基因型臨床分離菌株各1株,2株菌均對四種一線抗結(jié)核藥物敏感。應(yīng)用改良羅氏培養(yǎng)基培養(yǎng),收集菌體,采用CTAB法提取全基因組DNA；用Roche 454 GS FLX測序儀進(jìn)行全基因組高通量測序,測序結(jié)果使用DNA序列拼裝軟件進(jìn)行初始序列拼裝,得到測序重疊群(contigs),人工手動與參考序列進(jìn)行blast比對后,確定各個測序重疊群位置關(guān)系和首尾連接方式并預(yù)測測序未覆蓋區(qū)(gap區(qū))的大小Gap區(qū)和測序低值區(qū)域,經(jīng)特異性PCR擴(kuò)增后,使用ABI 3730測序儀進(jìn)行測序�；蚪M全長序列使用Phred、Phrap和Consed軟件包進(jìn)行拼裝和堿基修正,最終得到CCDC06029和CCDC06153兩株菌的全基因組序列數(shù)據(jù),與其他從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),在全基因組水平進(jìn)行比對、分析。結(jié)果測序得到中國結(jié)核分枝桿菌EAI和CAS基因型菌株CCDC06029(?)CCDC06153的全基因組完整序列,大小分別為4406422 bp和4411683 bp,其基因組測序數(shù)據(jù)的準(zhǔn)確度分別為99.9992%和99.9995%,預(yù)測的蛋白質(zhì)編碼序列(predicted protein coding sequences, CDSs)分別為4118個和4175個,CDSs平均大小分別為944bp和941bp。通過與已完成全基因組測序的結(jié)核分枝桿菌復(fù)合群菌株的全基因組數(shù)據(jù)的比對分析發(fā)現(xiàn)了存在于中國結(jié)核分枝桿菌EAI和CAS基因型中的多種分子特征。EAI基因型測序菌株CCDC06029異于結(jié)核分枝桿菌H37Rv的SNP位點(diǎn)共有2967個,其中位于蛋白編碼區(qū)的SNP有2650個；Indel數(shù)目為430個。CAS基因型測序菌株CCDC06153異于結(jié)核分枝桿菌H37Rv的SNP位點(diǎn)共有2213個,其中位于蛋白編碼區(qū)的SNP有1802個；Indel數(shù)目為374個。在此次研究中發(fā)現(xiàn)1011個EAI基因型特異性SNP,其中852個SNP位于蛋白編碼區(qū)內(nèi)；發(fā)現(xiàn)723個CAS基因型特異性SNP,其中547個位于蛋白編碼區(qū)內(nèi)。EAI基因型菌株CCDC06029與結(jié)核分枝桿菌標(biāo)準(zhǔn)菌株H37Rv比對分析后,共發(fā)現(xiàn)39個可能與毒力基因相關(guān)的SNP位置,其中非同義突變有20個,同義突變有19個。CAS基因型菌株CCDC06153與結(jié)核分枝桿菌標(biāo)準(zhǔn)菌株H37Rv比對分析后,共發(fā)現(xiàn)28個可能與毒力基因相關(guān)的SNP位置,其中非同義突變有13個,同義突變有15個。結(jié)論成功完成了中國結(jié)核分枝桿菌EAI和CAS基因型菌株的全基因組測序,為進(jìn)一步研究EAI和CAS基因型菌株的基因結(jié)構(gòu)特征、致病機(jī)制、分子進(jìn)化等提供了全基因組序列數(shù)據(jù)。全基因組數(shù)據(jù)的初步分析發(fā)現(xiàn)EAI和CAS基因型菌株基因組序列中存在某些特異性的Indel、SNP及其它分子生物學(xué)特征,將對EAI和CAS基因型的快速鑒定、溯源等多個方面的研究提供良好基礎(chǔ),并將促進(jìn)對結(jié)核分枝桿菌生物學(xué)本質(zhì)的認(rèn)識,對于有效防治結(jié)核病具有重要意義。
[Abstract]:Objective to sequence the whole genome of Mycobacterium tuberculosis EAI and CAS genotype clinical isolates (CCDC06029 and CCDC06153), and to compare and analyze the data of other Mycobacterium tuberculosis complex strains which have completed the whole genome sequencing, and to understand the whole genomic characteristics of the EAI and CAS genotypes, so as to further deepen the study of the genome of the strains of EAI and CAS. Studies, such as microevolution, toxic mechanics, etc., provide the basis. Methods 1 strains of Mycobacterium tuberculosis EAI and CAS genotypes were selected by bacteriology, drug sensitivity and molecular typing, and 2 strains were sensitive to four kinds of first-line anti tuberculosis drugs. The improved Roche culture medium was used to collect the mycelium and the whole base was extracted by CTAB method. The whole genome high throughput sequencing was carried out with the Roche 454 GS FLX sequencer. The sequencing results were sequenced using the DNA sequence assembly software for the initial sequence assembly, and the sequencing overlap group (contigs) was obtained. After the manual manual and the reference sequence was compared to the sequence, the location relationship and the first and the tail connection mode of the sequencing overlaps were determined and the sequencing was predicted. The size Gap area of the cover area (gap area) and the sequence of low value regions were amplified by specific PCR and were sequenced using ABI 3730 sequencer. The whole genome sequence used Phred, Phrap and Consed software packages to assemble and modify the base. Finally, the whole genome sequence data of the two strains of CCDC06029 and CCDC06153 were obtained, and the other from the NCBI genome web site. The whole genome data of the Mycobacterium tuberculosis complex strains were compared and analyzed at the whole genome level. The whole genome sequence of EAI and CAS genotype CCDC06029 (?) CCDC06153 of Mycobacterium tuberculosis in China was sequenced, the size of the genome was 4406422 BP and 4411683 BP respectively. The accuracy of the genome sequencing data was respectively For 99.9992% and 99.9995%, the predicted protein coding sequences (predicted protein coding sequences, CDSs) were 4118 and 4175 respectively. The average CDSs size of 944bp and 941bp., respectively, was found in Chinese tuberculosis through comparison and analysis of all genome data of the genome of Mycobacterium tuberculosis with complete genome sequencing. A variety of molecular characteristics in the EAI and CAS genotypes of Mycobacterium tuberculosis,.EAI genotypes, CCDC06029 are different from SNP loci of Mycobacterium tuberculosis H37Rv, of which there are 2650 SNP in the protein coding region; Indel number is 430.CAS genotype sequencing strains, CCDC06153 is different from the H37Rv SNP loci of Mycobacterium tuberculosis. There were 1802 SNP in the protein coding region and 374 in the Indel number. In this study, 1011 EAI genotype specific SNP were found, of which 852 SNP were located in the protein coding region; 723 CAS genotype specific SNP were found, and 547 of the.EAI genotype strains were located in the protein coding region, CCDC06029 and the standard Mycobacterium tuberculosis After H37Rv comparison, 39 SNP locations associated with virulence genes were found, of which there were 20 non synonymous mutations, and 19.CAS genotype strains, CCDC06153, and standard strains of Mycobacterium tuberculosis were compared and analyzed by the synonymous mutation, and a total of 28 possible locations of virulence genes were found, including 13 non synonymous mutations, and synonyms. There are 15 mutations. Conclusion the whole genome sequence of EAI and CAS genotypes of Mycobacterium tuberculosis in China has been successfully completed. The whole genome sequence data are provided for further research on the gene structure characteristics of EAI and CAS genotype strains, the pathogenesis mechanism and the molecular evolution. The preliminary analysis of the whole genome data has found the EAI and CAS genotypes. The existence of certain specific Indel, SNP and other molecular biological characteristics in the sequence of the group will provide a good basis for the rapid identification of EAI and CAS genotypes, traceability and so on, and will promote the understanding of the biological nature of Mycobacterium tuberculosis, which is of great significance for the effective prevention and treatment of tuberculosis.

【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】：2012
【分類號】：R378.911

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