【作者】 李丹；

【导师】 施汉昌； 何苗；

【作者基本信息】 清华大学 ， 环境科学与工程， 2010， 博士

【摘要】 全球范围内由病毒污染环境水体而引起疾病暴发事件不断发生,严重威胁着水质安全和人类健康,建立水环境中感染性病毒快速分析方法,综合分析水处理过程对病毒的去除特性,对预防控制病毒暴发、保障水质安全及防范健康风险具有重要的意义。本研究建立了轮状病毒(Rotavirus,RV)和腺病毒(Adenovirus,Ad)定量PCR(qPCR)方法,检测限约为102 copies/反应(约0.2 PFU/反应),定量区间为10~2-10~8 copies/反应;建立了细胞培养与qPCR相结合(ICC-RT-qPCR)方法测定感染性RV,最低检测限为0.2 PFU/mL,定量区间为0.2-200 PFU/mL(R~2>0.95);研究建立了利用荧光计数细胞(Fluorescence-activated cell sorting,FACS)方法快速检测感染性Ad,最低检测限为1 PFU/mL Ad,定量区间为1-10~4 PFU/mL(R~2 > 0.95)。ICC-RT-qPCR和FACS方法将传统空斑法检测感染性病毒的时间从7-14天缩短至2-3天,且灵敏性有所提高。采用ICC-RT-qPCR评价不同消毒方法效果结果表明,在紫外线剂量为360 mJ/cm~2和初始氯浓度为20 mg/L时(接触60 min),仍能检测出具有感染性的RV和其基因片段。由于该方法比空斑法灵敏,与空斑法评价结果相比,达到相同的灭活率,紫外线剂量需要由42 mJ/cm~2增加到117 mJ/cm2(4 log10),初始氯浓度由1 mg/L增加至5 mg/L(接触60 min)(1.7 log10)。不同初始氯浓度下,自由氯与病毒反应的主要部位不同,浓度低时主要是病毒衣壳蛋白,浓度高时主要是病毒核酸,浓度中等时,是两者共同与氯反应导致病毒灭活。研究中对北京市三个污水处理厂进出水进行一年(2007.05-2008.04)的监测。结果表明,污水中RV秋冬季浓度高,夏季浓度低,污水一级和二级处理出水中感染性RV冬季平均浓度分别为:288-689 PFU/L和0.6-2.9 PFU/L;不同处理工艺对RV的去除率从高到低依次是:二级处理工艺:A~2/O(2.65-2.83 log10)>传统活性污泥法(2.22 log10),三级处理工艺:反渗透(1.29 log10)>膜过滤(0.91 log10)>砂滤系统(0.78 log10)。本研究在雨季对美国洛杉矶Segundo海湾某海水淡化厂进出水进行检测,结果表明降雨对海水中Ad污染无明显影响。更多还原

【Abstract】 Waterborne viral infection is one of the most important causes of human morbidity and mortality, and epidemics associated with waterborne viruses are well documented worldwide. Therefore, it’s urgent to develop rapid and sensitive detection methods in order to determine the concentration profiles of waterborne viruses in the environments, and to evaluate the efficiency of viral removal and inactivation during wastewater treatment processes.Quantitative PCR (qPCR) for rapid detection of rotaviruses (RV) and adenoviruses (RV) were established, the detection limits are approximately 102 copies /reaction, and the quantitative ranges are from 10~2 to 108 copies /reaction; An integrated cell culture and qPCR (ICC-RT-qPCR) was developed to detect infectious RV, which can detect as few as 0.2 PFU/mL, a linear correlation was obtained by plotting RV copies detected by ICC-RT-qPCR versus the logarithm of initial RV inoculated at concentrations ranging from 0.2 to 200 PFU/mL. In addition, a fluorescence-activated cells sorting (FACS) was established to rapidly and sensitively determine and quantify infectious Ad, with a detection limit of 1 PFU/mL and a linear correlation obtained by plotting the percentages of hexon-positive cells versus the logarithm of initial Ads inoculated ranging from 1 to 104 PFU/mL. Both the ICC-RT-qPCR and FACS assays reduced the detection time of infectious virus from 7-14 day by conventional plaque assay to 2-3 days with better sensitivities.The newly developed ICC-RT-qPCR method was applied to investigate the inactivation and resistance of RV to UV and free chlorine disinfection, and to compare the results with those obtained by conventional assays. The results showed that RV evaluated by ICC-RT-qPCR was more resistant to UV and free chlorine disinfection than that evaluated by plaque assay. Infectious RV and gene segments were detectable at UV dose of 360 mJ/cm2 and chlorine dose of 1200 mg·min/L. To achieve the same removal efficiency evaluated by plaque assay, the required UV dose and chlorine dose evaluated by ICC-RT-qPCR increased from 42 to 117 mJ/cm2 (4 log10) and from 60 to 300 mg·min/L (1.7 log10), respectively. The key target of RV during chlorine disinfection varies with different initial free chlorine concentrations. The damage of outside protein is pivotal target at low concentration, while the damage of nucleic acid is crucial at high concentration, and both the damages of protein and nucleic acid result in the inactivation of rotavirus at moderate concentration of free chlorine.The contaminations of waterborne viruses may be impacted by the epidemics, urban runoff and the waterbody quality. A one-year monthly monitoring of RV in raw and treated wastewater from three typical municipal wastewater treatment plants (WWTPs) in Beijing of China was conducted. Results showed that RVs were abundant in autumn and winter but rare in summer. During autumn and winter seasons, the average removal efficiencies of infectious RV in primary and secondary treated wastewater were 288-689 and 0.6-2.9 PFU/L, respectively. Among the different types of secondary and tertiary treatment processes, the anaerobic-anoxic-oxic (A2/O) process had higher removal efficiency than conventional activated sludge process, and the reverse osmosis (RO) was more efficient to remove infectious RV than membrane ultrafiltration and conventional flocculation-sedimentation and sand filtration process. The viral quantity of seawater intaked into the desalination plant at West Basin Desalination Plant at El Segundo, Los Angeles of USA, were evaluated during periods of coastal storms, which are used for drinking water production by reverse osmosis (RO). The results indicated that the adenoviral contamination in the seawater intake was not significantly impacted by the storm or urban runoff.更多还原