结核分枝杆菌基因组学及特异检测技术研究

IOCAS-IR > 海洋环流与波动重点实验室

	结核分枝杆菌基因组学及特异检测技术研究
其他题名	Research on genomics and specific detection technique of Mycobacterium Tuberculosis
	周厚清
学位类型	博士
	2009-05-13
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	基因组结核分枝杆菌实时聚合酶联反应
摘要	结核分枝杆菌(M.tuberculosis)，俗称结核杆菌，是引起结核病的病原菌。可侵犯全身各器官，但以肺结核为最多见。结核病至今仍为重要的传染病。估计世界人口中1/3感染结核分枝杆菌。据WHO报道，每年约有800万新病例发生，至少有300万人死于该病。我国建国前死亡率达200-300人/10万，居各种疾病死亡原因之首，建国后，随着人民生活水平提高，卫生状态改善，特别是开展了群防群治，儿童普遍接种卡介苗，结核病的发病率和死亡率大为降低。由于耐药结核的出现已及HIV共感染等因素影响，目前发病率又有上升趋势。对于结核病的防治而言，其首要问题是结核病的诊断，而结核病的实验室检查已成为临床医生诊断结核病、判断疗效和评估预后不可缺少的手段。结核病的临床诊断主要根据病史、痰培养、涂片抗酸染色、胸片等结果，其中痰结核检查是诊断肺结核的重要依据之一，检测方法常规采用细菌学检查。痰中细菌学检查主要采用痰涂片和培养方法，涂片虽简单易行，但阳性率低，培养虽为金标准，但周期太长，均难以满足临床需要。细菌对部分患者可造成误诊或漏诊。近年来随着现代科学技术的不断进展，分子生物学技术在结核病诊断方面取得了显著的成绩，实时动态荧光定量(FQ—PCR)技术又是在原有的PCR技术上一次质的飞跃。尽管许多文献已经报道了结核耐药的分子机理，但耐药的分子机理并没有充分了解。本研究利用全基因组鸟枪法测序和高通量测序技术，测定了两株结核分枝杆菌菌株TFJA和TFJB全基因组序列，分别为药物敏感株和耐多药株。在序列初步拼接后，获得了由189个DNA contigs（DNA序列连接群）组成的基因组框架图。借助10kb左右插入片段克隆的正反向末端序列信息以及PCR扩增技术，确定了全部DNA contigs在基因组上的物理位置，最后补上了所有“间隙(gap)” ，得到了全基因组序列。三种限制性酶切物理图谱证明了序列拼接的正确性。两株结核菌基因组大小为4.4Mbp左右，GC含量都在65.6%左右，其中TFJB株比TFJA株大7kbp，分别具有3591、3648个基因，83.7%的基因位于先导链上。我们比较了两株细菌的949SNPs位点，发现94个在TFJB中不同nsSNPs，这些SNPs位点的碱基在其它的结核分枝杆菌基因组中是相同的。我们去掉了那些代谢途径与毒力代谢无关的基因，最后选择了6个基因的18个SNP，检测这些SNP在277株耐药或者药物敏感结核分枝杆菌中的保守性以及其耐药相关性。用PCR扩增含有SNP的序列，用统计学检验这些SNP是否耐药相关，结果发现，TFJB0109_CDS04405基因的407位点SNP（g->a）和TFJB0109_CDS00149基因上的SNP（720a->c，722插入t，727t->g，729g->t，以及735g->t）可能与SM耐药相关，其P-value分别是0.04128、0.02160和0.01394（见表3）。结核是一种慢性传染病，其病原体为TB。在全基因组序列分析研究的同时，我们筛选到一套灵敏度较高、特异性较好的REAL TIME-PCR检测引物，并用它对105份不同发病时间的病人标本进行了检测。初步结果表明，应用本方法，样本检出率为98%，这一结果比涂片及分离培养更灵敏。
其他摘要	Tuberculosis (TB) is contagious and spreads through the air. It is one of the leading causes of infectious disease mortality in the world, with over 2 million deaths recorded annually, and it is estimated that one third of the world's population is latently infected. One in every 10 of those people will become sick with active TB in his or her lifetime. If not treated, each person with active TB can infect on average 10 to 15 people a year. And a total of 1.6 million people died from TB in 2005, equal to about 4400 deaths a day. Strains that are resistance to a single drug have been documented in every country surveyed; what is more, strains of TB resistance to all major anti-TB drugs have emerged, and a particularly dangerous form of drug-resistance TB is multidrug-resistance TB (MDR-TB), which is defined as the disease caused by TB bacilli resistance to at least isoniazid and rifampicin, the two most powerful anti-TB drugs. Rates of MDR-TB are high in some countries, especially in the former Soviet Union, and threaten TB control efforts. In this study, we determined the whole genome sequence of the M. tuberculosis drug-resistance strains TFJB. Genome comparison of TFJB and other six sensitive strains which published on NCBI to find that there may be some other drug-resistance site in TB, except known drug-resistance genes. TFJB was 4,405,981 bps, 5kbps shorter than sequenced genome H37Rv, with 65.6% GC content. 2.04% repeat regions of genome (~89K) are found with repeat mask. We found 3591 genes and one set 16s-23s-5s ribosome RNA and 45 tRNA in the genome. Tuberculosis (TB) is a major cause of illness and death worldwide, especially in Asia and Africa. Rapid detection of TB is very useful for patient management as well as epidemics control. Nucleic acid amplification-based techniques (NAT) have become an important tool for TB diagnosis and posses enormous advantage of providing a result within hours. The real-time PCR assay has many advantages over traditional NAT-based assays including rapidity, quantitative measurement, low contamination rate, higher sensitivity, higher specificity, and easy standardization. In this study, we presented a rapid, specific and sensitive assay for the identification the TB by using the real-time PCR assay. The probe was specific to the IS6110 gene region. The linear range of the TB was widely which was from 10 copies/reaction to 4×107copies/reaction (r2=0.9959). To determine the specificity of the real-time PCR assays, 60 sputum samples of healthy people were tested. The assays reacted negatively to all. To determine the sensitivity of the real-time PCR assays, 105 sputum samples of patients were tested. 104 samples tested positive. Compared to Smear microscopy (36/105) and Mycobacterial culture (41/105), real-time PCR is very fast and sensitivity.
页数	67
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1029
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	周厚清. 结核分枝杆菌基因组学及特异检测技术研究[D]. 海洋研究所. 中国科学院海洋研究所,2009.

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