宏基因组学技术在病原体检测中的应用

发布时间：2019-01-08 18:11

【摘要】：环境微生物是地球上种类最多、数量最大、分布最广的生物类型,据估计,地球上存在超过1030个微生物。微生物的传统研究方法主要是基于微生物纯培养和分离,到目前为止,绝大多数微生物(99%以上)无法依靠上述方法获得,这极大地限制了对微生物的研究。借助于高通量测序技术快速发展起来的宏基因组学,在病原体检测中发挥着越来越重要的作用。宏基因组测序无需筛选得到各微生物群落的纯培养物,而是直接测定样品中所有微生物的核酸序列,这样可避免实验污染带来的偏差。宏基因组学的研究主要有16S rDNA测序和全基因组测序两种。16S rDNA测序以环境样品中的16S rDNA为研究对象,全基因组测序(whole genome sequencing)只需直接提取出微生物基因组进行测序。本研究基于宏基因组测序,探索未知病因样本中可能存在的致病微生物,为揭示疾病的发生和发展提供线索。我们提取10份烂脚病样品细菌核酸,扩增16S rDNA的V1~V2高变区,测序后分析其中的菌群构成,在所有样本中均有检出与伤口溃烂有关的金黄色葡萄球菌和链球菌。16S rDNA测序有较多环节需要改进,如细菌核酸的提取、高变区的选择、通用引物的选择、测序平台的选择以及后期数据分析策略的选择。我们试验了七种方法对模拟样本进行细菌核酸提取,测序后,分析模拟样本中各种细菌的含量和相对分布,寻找一种广谱、高效的细菌核酸提取方法。在筛查一份未知病因发热病人的样品时,我们测序了其中病毒的宏基因组,发现了一株属于Microviridae的病毒基因组IME-16,与以往报道差异比较大,我们对其进行了注释、进化分析以及结构蛋白的分析,推测IME-16可能是与alphaviruses距离较远的一支。在测序一份乙肝病人血清样品的病毒宏基因组时,我们优化了生物信息分析方法,加快了数据分析的速度,同时也发现了一些以往报道与肝炎有关的Anelloviridae病毒序列,其中共发现10株Torque Teno Midi Virus(TTMDV)序列和3株Torque Teno Virus(TTV)序列与以往报道的序列差异大,我们对这些序列进行注释和进化分析,证实它们属于新型的Anelloviridae病毒。综上所述,我们采用宏基因组测序,筛查了一批未知病因烂脚病样品、一份未知病因发热血清样品以及一份乙型肝炎病人血清样品,发现了与疾病有关的致病菌以及新型病毒全基因组序列,同时,我们改进细菌基因组提取方法和数据分析方法。总之,随着宏基因组测序方法改进和分析技术提高,宏基因组学必将在病原体检测方面作出重大贡献。
[Abstract]:Environmental microorganisms are the most abundant and widely distributed organisms on the earth. It is estimated that there are more than 1030 microbes on the earth. The traditional research methods of microorganisms are mainly based on the pure culture and isolation of microorganisms. Up to now, most of the microorganisms (over 99%) cannot be obtained by these methods, which greatly limits the research of microbes. Macrogenomics, which is rapidly developed by high throughput sequencing, plays a more and more important role in pathogen detection. The nucleic acid sequences of all microbes in the samples can be determined directly without screening the pure culture of each microbial community by macro genome sequencing, which can avoid the deviation caused by the experimental contamination. The study of macrogenomics mainly includes 16s rDNA sequencing and whole genome sequencing. 16s rDNA sequencing takes 16s rDNA in environmental samples as the research object. The whole genome sequencing (whole genome sequencing) only need to extract the microbial genome directly for sequencing. Based on macro genome sequencing, this study explored the possible pathogenic microorganisms in the samples of unknown etiology and provided clues for the occurrence and development of the disease. We extracted bacterial nucleic acid from 10 samples of rotting foot disease and amplified the V1~V2 hypervariable region of 16s rDNA. Staphylococcus aureus and Streptococcus streptococcus associated with wound ulceration were detected in all samples. 16s rDNA sequencing needs to be improved, such as the extraction of bacterial nucleic acid, the selection of hypervariable region, and the selection of universal primers. The selection of sequencing platform and later data analysis strategy. We tested seven methods to extract bacterial nucleic acid from simulated samples. After sequencing, we analyzed the content and relative distribution of various bacteria in simulated samples, and looked for a broad spectrum and high efficiency method for bacterial nucleic acid extraction. In screening a sample of a patient with an unknown cause of fever, we sequenced the macro genome of the virus, and found that the genome IME-16, of a virus belonging to Microviridae was quite different from previous reports, and we annotated it. Evolution analysis and structural protein analysis suggested that IME-16 might be a branch far away from alphaviruses. When sequencing the virus macro genome of a hepatitis B patient's serum sample, we optimized the analysis method of biological information, accelerated the speed of data analysis, and found some previously reported Anelloviridae virus sequences related to hepatitis. A total of 10 Torque Teno Midi Virus (TTMDV) sequences and 3 Torque Teno Virus (TTV) sequences were found to be quite different from those previously reported. We annotated and analyzed these sequences and confirmed that they belong to a new type of Anelloviridae virus. To sum up, we used macro genome sequencing to screen a batch of samples for unknown causes of foot rot, an unknown cause of fever, and a sample for hepatitis B patients. The whole genome sequences of pathogenic bacteria and new viruses have been found. Meanwhile, we have improved the methods of bacterial genome extraction and data analysis. In a word, with the improvement of the method of macro genome sequencing and the improvement of analysis technology, macrogenomics will make a great contribution to the detection of pathogens.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5

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