水环境中三种病毒PMA-PCR方法建立与基于焦磷酸测序技术的细菌多样性研究
发布时间：2018-11-24 09:25
【摘要】：目前,我国最新颁布的饮用水相关标准有六项微生物指标,无法全面地反映水中病原微生物的污染状况,还缺少易引起腹泄、肠胃炎等肠道疾病且一般氯消毒方法很难全部杀死的病毒类指标。因此,有必要开展病毒类病原微生物的研究和监测工作。本论文首次建立了PMA-PCR方法用于定性检测活性的轮状病毒、星状病毒和腺病毒,同时也首次建立了PMA-qPCR方法用于定量检测活性的轮状病毒,并将这些方法应用于筛查重点流域典型水源地相关病毒的污染情况。实验结果显示利用PMA-PCR法检测加热处理和未加热处理的阳性样本时表现出显著差异,表明该方法能够区分活性和非活性的轮状病毒、星状病毒、腺病毒。实验结果也显示利用PMA-qPCR法检测加热处理和未加热处理的轮状病毒阳性样本时表现出显著差异,表明该方法能够区分活性和非活性的轮状病毒。这些研究结果证明了PMA-PCR法、PMA-qPCR法的可靠性。在重点流域典型水源地水样的实际应用检测中,辽河流域的QH采样点有活性轮状病毒检出。经PMA-qPCR法定量测定后,该采样点活性轮状病毒的浓度为1.24×105Copies/L研究结果充分证明了PMA-PCR法能够成功地应用于三种活性病毒的检测,也充分证明了PMA-qPCR法能够成功地应用于轮状病毒的定量检测。 另一方面,为了减轻城市用水压力和美化环境,大部分北京地表水都接纳了污水处理厂的出水。目前,有研究报道中国的14个污水处理厂的活性污泥样本存在核心菌群和独有菌群,而接纳污水处理厂出水的北京地表水的细菌群落情况仍未见有报道,因此有必要调查分析北京地表水细菌群落的情况。本研究首次利用焦磷酸测序技术分析5个北京地表水水样的细菌多样性,作为初步监测复杂环境中细菌群落的基本参考。经过测序得到60810条优化序列,平均长度为400bp,并使用Mothur软件对优化序列进行归类。研究结果充分展示了从门水平到属水平的地表水菌群组成情况,其中相对丰度较高的门依次为变形菌门(Proteobacteria),其相对丰度分别占总OTU数目的20.28%～67.89%；放线菌门(Actinobacteria),分别占总OTU数目的19.78%～53.59%；拟杆菌(Bacteroidetes),分别占总OTU数目的3.85%～16.14%：另外,还有疣微菌门(Verrucomicrobia)和蓝藻门(Cyanobacteria),分别占总OTU数目的0.55%-7.25%和0.68%-2.60%等。这5个门分别占总OTU数目91.70%-98.50%。研究结果还显示了5个北京地表水样本中存在核心菌群,即门水平上有13个核心菌群,纲水平上由23个核心菌群,目水平上有32个核心菌群,科水平上有40个核心菌群,属水平上有40个核心菌群。还证明了5个处于不同地理位置的地表水样中存在相对丰度0.5%的独有菌群,即目水平上有19个独有菌群,科水平上有49个独有菌群,属水平上有100个独有菌群。通过Weighted Unifrac PCA分析表明处于地表水样本1和样本2的细菌群落结构更相似。此外,研究结果还说明了污水处理厂活性污泥样本的细菌群落多样性对北京地表水的细菌群落多样性有一定的影响。
[Abstract]:At present, the newly-issued drinking water-related standard in China has six microbial indicators, which can not fully reflect the pollution condition of pathogenic microorganisms in water, but also lacks the virus type indexes which are easy to cause intestinal diseases such as abdominal diarrhea and gastroenteritis, and the general chlorine disinfection method is difficult to kill all. Therefore, it is necessary to carry out the research and monitoring of the virus-like pathogenic microorganisms. The first time in this paper, a PMA-PCR method was established for the qualitative detection of the active rotavirus, star-like virus and adenovirus, and the PMA-qPCR method was also first established for the quantitative detection of the active rotavirus, and these methods were used to screen the contamination of the typical water source-related virus in the key watershed. The results show that the method can distinguish the active and inactive rotavirus, star-like virus and adenovirus by using the PMA-PCR method to detect the positive samples of the heat treatment and the unheated treatment. The results of the experiments also showed a significant difference in the detection of the positive samples of rotavirus by the PMA-qPCR method and the untreated rotavirus, indicating that the method was able to distinguish between active and inactive rotavirus. The results of these studies demonstrated the reliability of PMA-PCR and PMA-qPCR. In the actual application of water sample in the typical water source of the key drainage area, the QH sampling point of the Liaohe river basin is detected by the active rotavirus. The results of PMA-qPCR method show that the concentration of the active rotavirus is 1. 24-105Copies/ L. The results of the study show that the PMA-PCR method can be successfully applied to the detection of three kinds of active viruses, and it is also proved that the PMA-qPCR method can be successfully applied to the quantitative detection of rotavirus. On the other hand, in order to reduce the urban water pressure and the beautifying environment, most of the surface water in Beijing has accepted the sewage treatment plant. At present, there is a study on the presence of the core bacteria and the unique flora in the activated sludge samples of the 14 sewage treatment plants in China, while the bacterial community of the Beijing surface water receiving the effluent from the WWTP is still not reported. Therefore, it is necessary to investigate and analyze the bacterial community of surface water in Beijing. In this study, the bacterial diversity of five Beijing surface water samples was analyzed by pyrosequencing technology for the first time, which was used as the basis for preliminary monitoring of the bacterial community in complex environment. Reference. The 60810 optimized sequence was obtained by sequencing, with an average length of 400bp, and the optimized sequence was performed using the Moothur software. The results of the study fully show the composition of the surface water species from the level of the door to the genus. The relative abundance of the relative abundance is 20. 28% to 67. 89% of the total OTU, and 19. 78% to 53. 5, respectively, of the total OTU. Bacteroides (9%) and Bacteroides, respectively, accounted for 3.85% ~ 16.14% of the total OTU. In addition, the number of total OTU was 0.55%-7.25% and 0.68%-2.60, respectively.%, etc. The 5 doors account for the total OTU number of 91.70% -98,5, respectively. The results of the study also show that there are 13 core strains in the five Beijing surface water samples, i.e., there are 13 core bacteria in the level of the phylum, and there are 32 core bacteria in the level. There are 40 core bacteria in the family level, and there are 40 cores in the level. There are also 5 unique bacterial groups with relative abundance of 0. 5% in surface water samples with different geographical locations. The bacterial population of surface water samples 1 and 2 was analyzed by the weighted Unifrac PCA analysis. Similarly, the results of the study also show that the diversity of the bacterial community in the activated sludge sample of the sewage treatment plant has a certain effect on the bacterial community diversity of the surface water in Beijing.
【学位授予单位】：北京林业大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R123.1;X172
本文编号：2353133
[Abstract]:At present, the newly-issued drinking water-related standard in China has six microbial indicators, which can not fully reflect the pollution condition of pathogenic microorganisms in water, but also lacks the virus type indexes which are easy to cause intestinal diseases such as abdominal diarrhea and gastroenteritis, and the general chlorine disinfection method is difficult to kill all. Therefore, it is necessary to carry out the research and monitoring of the virus-like pathogenic microorganisms. The first time in this paper, a PMA-PCR method was established for the qualitative detection of the active rotavirus, star-like virus and adenovirus, and the PMA-qPCR method was also first established for the quantitative detection of the active rotavirus, and these methods were used to screen the contamination of the typical water source-related virus in the key watershed. The results show that the method can distinguish the active and inactive rotavirus, star-like virus and adenovirus by using the PMA-PCR method to detect the positive samples of the heat treatment and the unheated treatment. The results of the experiments also showed a significant difference in the detection of the positive samples of rotavirus by the PMA-qPCR method and the untreated rotavirus, indicating that the method was able to distinguish between active and inactive rotavirus. The results of these studies demonstrated the reliability of PMA-PCR and PMA-qPCR. In the actual application of water sample in the typical water source of the key drainage area, the QH sampling point of the Liaohe river basin is detected by the active rotavirus. The results of PMA-qPCR method show that the concentration of the active rotavirus is 1. 24-105Copies/ L. The results of the study show that the PMA-PCR method can be successfully applied to the detection of three kinds of active viruses, and it is also proved that the PMA-qPCR method can be successfully applied to the quantitative detection of rotavirus. On the other hand, in order to reduce the urban water pressure and the beautifying environment, most of the surface water in Beijing has accepted the sewage treatment plant. At present, there is a study on the presence of the core bacteria and the unique flora in the activated sludge samples of the 14 sewage treatment plants in China, while the bacterial community of the Beijing surface water receiving the effluent from the WWTP is still not reported. Therefore, it is necessary to investigate and analyze the bacterial community of surface water in Beijing. In this study, the bacterial diversity of five Beijing surface water samples was analyzed by pyrosequencing technology for the first time, which was used as the basis for preliminary monitoring of the bacterial community in complex environment. Reference. The 60810 optimized sequence was obtained by sequencing, with an average length of 400bp, and the optimized sequence was performed using the Moothur software. The results of the study fully show the composition of the surface water species from the level of the door to the genus. The relative abundance of the relative abundance is 20. 28% to 67. 89% of the total OTU, and 19. 78% to 53. 5, respectively, of the total OTU. Bacteroides (9%) and Bacteroides, respectively, accounted for 3.85% ~ 16.14% of the total OTU. In addition, the number of total OTU was 0.55%-7.25% and 0.68%-2.60, respectively.%, etc. The 5 doors account for the total OTU number of 91.70% -98,5, respectively. The results of the study also show that there are 13 core strains in the five Beijing surface water samples, i.e., there are 13 core bacteria in the level of the phylum, and there are 32 core bacteria in the level. There are 40 core bacteria in the family level, and there are 40 cores in the level. There are also 5 unique bacterial groups with relative abundance of 0. 5% in surface water samples with different geographical locations. The bacterial population of surface water samples 1 and 2 was analyzed by the weighted Unifrac PCA analysis. Similarly, the results of the study also show that the diversity of the bacterial community in the activated sludge sample of the sewage treatment plant has a certain effect on the bacterial community diversity of the surface water in Beijing.
【学位授予单位】：北京林业大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R123.1;X172
【参考文献】
相关硕士学位论文 前3条
1 张德友;北京地区水环境中轮状病毒的分布规律及其健康风险评价[D];北京林业大学;2009年
2 张晓红;北京地区成人腹泻诺如病毒的流行和分子特征的初步研究[D];中国疾病预防控制中心;2009年
3 华蔚颖;应用454测序技术分析菌群结构的方法学研究[D];上海交通大学;2010年
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