【作者】 张崇淼；

【导师】 王晓昌；

【作者基本信息】 西安建筑科技大学 ， 环境工程， 2008， 博士

【摘要】 水环境的病原性污染是当今世界上危害最严重的问题之一。研究水体病原性污染状况，分析病原体对人类健康造成的风险具有十分重要的意义。用传统的微生物指标评价病原体造成的健康风险具有多方面的不确定性，而近年来发展起来的PCR技术具有灵敏度高、特异性好等优点，已在医学及相关领域的病原体检测中体现出其优越性。但是目前PCR技术用于水环境病原体检测还处于尝试阶段，有效的方法体系尚未建立，以此为基础的病原体健康风险评价方法还是空白。为此，本论文在国家自然科学基金项目“水环境中肠道感染性病毒群的PCR综合鉴定和病原性污染风险评价”的支持下开展研究，建立了高效实用的肠道病原体PCR检测方法和病原性污染评价指标，在进行水环境长期监测的基础上研究了肠道病原体在地表水体中的分布和变化规律，基于肠道病原体PCR检测的结果对不同水体进行了健康风险评价。本论文根据我国水传播疾病和水体病原性污染的特点，选取肠道病毒作为典型的病毒性病原体，伤寒沙门菌、志贺菌、大肠埃希菌和霍乱弧菌作为典型的肠道病原菌，分别建立了定性和定量的PCR检测方法。采用改进的微孔滤膜吸附-洗脱浓缩法进行水体样品中肠道病毒的浓缩，研究了滤膜的孔径、材质、洗脱剂的种类、洗脱方式、PEG的浓度对病毒回收率的影响，进行了病毒浓缩方法的优化。研究结果表明，0．22μm孔径的混合纤维素酯微孔滤膜比硝酸纤维素和尼龙膜的实用效果更好；采用3％牛肉膏-0．05mol／L甘氨酸缓冲液作为洗脱剂，使用磁力搅拌方式进行膜洗脱的方法具有效果好，操作方便等优点；在洗脱液浓缩步骤中，当PEG的最终浓度为130g／L时，病毒的回收率最高；改进的微孔滤膜吸附-洗脱法的病毒回收效果优于滑石粉-硅藻土吸附层吸附-洗脱法、化学絮凝沉淀法和滑石粉-硅藻土絮凝沉淀法等方法，对于不同接种量(38～3800CCID₅₀)的病毒回收率均可达到70％以上。根据肠道病毒RNA5′非编码区中保守序列的同源性设计了针对首轮PCR和巢式PCR的两对肠道病毒通用引物，经GenBank数据库BLAST比对分析，所设计的通用引物涵盖了脊髓灰质炎病毒、柯萨奇病毒、埃可病毒、肠道病毒71型等多种肠道病毒。利用脊髓灰质炎病毒1～3型，柯萨奇病毒B3型作为参考病毒株，建立了肠道病毒巢式RT-PCR检测方法。结果表明，AMV酶能够有效完成病毒RNA的逆转录，比M-MLV酶更具有适用性；镁离子浓度2mmol／L，退火温度55℃是最佳的首轮PCR反应条件；巢式PCR不仅具有良好的特异性，而且灵敏度高达0．038CCID₅₀，适用于低病毒浓度的环境水样检测。在肠道病毒RT-PCR检测的基础上，建立了实时荧光定量RT-PCR检测方法，利用pMD18-T载体构建了重组质粒作为标准品，确定了反应体系中镁离子的最佳浓度为4．0mmol／L，最佳退火温度为55℃，读板温度为85℃；检测灵敏度为4．62GEC，且特异性良好，与其他微生物没有交叉反应；动力学线性范围广，在4．62～4．62×10⁹GEC的模板量范围内，模板量的对数值与C_T值之间具有良好的线性关系（R²=0．997）；精密度高，检测样品C_T值批内变异系数低于2％，批间变异系数低于5％。针对典型肠道病原菌，分别建立了伤寒沙门菌、志贺菌、大肠埃希菌和霍乱弧菌的常规PCR检测和实时荧光定量PCR检测方法。利用标准菌株，建立了典型肠道病原菌的实时荧光定量PCR标准曲线，伤寒沙门菌、志贺菌和大肠埃希菌的检测灵敏度分别为0．366CFU、0．44CFU和4．56CFU；当模板量分别在0．366CFU～3．66×10⁵CFU，0．44CFU～4．4×10⁵CFU和4．56CFU～4．56×10⁵CFU的范围内时，模板量的对数值与C_T值之间具有良好的线性关系。运用本研究建立的PCR检测方法，对西安市典型地表水体（饮用水源水—黑河）、景观湖泊—兴庆湖和北湖、城市排污河—浐河)中的肠道病原体进行了长期监测，并以污水处理厂二级处理出水作为参比，研究了肠道病毒和典型肠道病原菌在水环境中的分布规律。长达1年的监测结果表明，肠道病毒在各地表水体和二级处理出水中均被检出，且检出浓度服从对数正态分布规律；黑河、兴庆湖、北湖、浐河和二级处理出水中肠道病毒的年平均浓度分别为47GEC／L、127GEC／L、120GEC／L、422GEC／L和746GEC/L；黑河的肠道病毒在春夏两季阳性率高，浓度也较高；兴庆湖和北湖的肠道病毒的高阳性率和高浓度发生在夏末秋初；浐河的肠道病毒的阳性率和浓度普遍偏高，且浓度变化幅度大；对参比的二级处理出水而言，9月初至12月中旬和4月下旬至5月末是肠道病毒的高浓度期。各水体中肠道病毒的核酸序列具有较强的相似性，表明病毒种类比较单一。在对伤寒沙门菌、志贺菌、大肠埃希菌和霍乱弧菌进行定量PCR检测的同时，也运用传统的培养法进行了细菌总数、大肠菌群的粪大肠菌群的检测。结果表明，霍乱弧菌在各地表水体中均呈阴性；其他各类病菌的检出浓度均服从对数正态分布规律；黑河、兴庆湖和北湖的伤寒沙门菌PCR检测结果全部为阴性，而浐河和二级处理出水的伤寒沙门菌浓度较高，但没有明显的季节变化规律；志贺菌在各种水体中均存在，但夏季之前浓度很低，进入夏季后浓度迅速升高；各水体的大肠埃希菌浓度都呈现从冬季到夏季逐渐升高的趋势。肠道病毒、典型肠道病原菌与传统细菌学和理化指标之间关系的分析结果表明：肠道病毒与细菌指标之间无显著相关性，在细菌浓度较低的水体中仍可能检出肠道病毒，细菌学指标难以代替肠道病毒的检测；水中细菌总数、大肠菌群、粪大肠菌群与水温、浊度、COD之间呈显著正相关，与溶解氧呈显著负相关；伤寒沙门菌的突增通常发生在大肠菌群、粪大肠菌群浓度超过10³CFU／100mL时；志贺菌与传统细菌学指标呈显著正相关，其阳性率和浓度随着大肠菌群和粪大肠菌群浓度增大而逐渐上升，在大肠菌群浓度较低的水体中仍有志贺菌出现。根据肠道病原体的PCR检测结果，结合暴露途径和剂量反应关系分析，运用数学模型对不同地表水体中肠道病原体造成的健康风险进行了评价。判定了各水体中的“最大风险肠道病原体”，并提出了相应的风险控制需求。评价结果表明：作为饮用水源的黑河水，肠道病毒是具有最大风险的肠道病原体，以10^-4／a作为可接受年风险，在肠道病毒去除率为6-log的条件下，可以保障水质安全性大于90％。作为景观娱乐水体的兴庆湖和北湖，肠道病毒仍是最大风险肠道病原体，当肠道病毒的去除率达到4-log时，水质安全性在90％以上。浐河的肠道病原体风险的主要来源是多方面的，若直接作为景观用水，则志贺菌和大肠埃希菌造成的健康风险高于肠道病毒，但由于肠道病原菌与肠道病毒分布的差异，当它们的去除率都为4-log时，与肠道病毒相关的水质安全性仍然较低。更多还原

【Abstract】 Pathogenic pollution of water environment is one of the most serious problemsall over the world.Study on pathogenic pollution of water environment and healthrisk assessment is of great significance.Many uncertainty factors in health risk ofpathogens using traditional pathogenic microorganism indicator,polymerase chainreaction （PCR）with advantages of high sensitivity and specificity has showedsuperiority in detection of pathogens in medical science.However,the research aboutusing PCR to detection pathogens in water environment is limited at present.There islack of effective PCR detection method,and the study of health risk assessment on thebasis of PCR result is blank.Therefore,funded by the National Natural ScienceFoundation of China project "Study on detection of enteric viruses and pathogenicrisk assessment",this study was carried out to establish effective PCR detectionmethod of pathogens,to investigate the distribution and variation of pathogens presentin urban surface water and to characterize the human health risk associated withpathogens based on the PCR results.According to the characteristics of waterborne diseases and pathogenic pollutionin water environment in our country,enteroviruses were selected as typical entericviruses;Salmonella typhi,Shigella,Escherichia coli and Vibrio cholerae wereselected as typical pathogenic bacteria in water environment in this study.Thequalitative PCR and real-time fluorescent quantitative PCR detection method of thetypical pathogens were established.Using microporous filter adsorption-elution method to concentrate enterovirusesin water samples,the effects of many factors including nominal pore size,material offilter,the type of eluant and elution,and PEG concentration on virus recovery wereinvestigated and the concentration method was optimized.The results showed that cellulose mixed-ester microporous filter with nominal pore size of 0.22μm was superior to cellulose nitrate filter and nylon filter.Magnetism agitation was used for membrane elution and the optimal final mass concentration of PEG was 130g/L ineluant concentration step.Compared with talcum powder-diatomaceous earth layeradsorption-elution method,chemical flocculation precipitation method and talcumpowder-diatomaceous earth flocculation precipitation method,the modifiedmicroporous filter adsorption-elution method established in this study had more than70 percent of virus recovery in various virus inoculations(38～3800CCID₅₀).Two pairs of universal primers of enteroviruses were designed by analyzing thehomology of the highly conserved 5’noncoding region nucleotide sequences of theenterovirus genome.Poliovirus type 1～3,Coxsackie virus type B3 were used asreference virus strains,RT-nested PCR method of enteroviruses was established.Compared with M-MLV transcriptase,AMV transcriptase was more suitable to thepractical application because both in surface water and sewage virus RNA all can betranscribed by AMV transcriptase.55℃annealing temperature and 2mmol/Lmagnesium ion was determined as optimized condition in first round PCR.ThisRT-nested PCR method had good specificity and high sensitivity (0.039CCID₅₀),thusit was more suitable for detecting low concentrations of virus in environmental watersamples.Based on the RT-PCR of enteroviruses,real-time fluorescent quantitativePCR of enteroviruses was established.The recombinant plasmid was constructed asenterovirus DNA standard by cloning poliovirus cDNA into a pMD18-T vector.Optimized concentration of magnesium ion was as 4.0mmol/L,annealing temperatureas 55℃,and reading plate temperature as 85℃.The results showed that the detectionlimit of 4.62 genome equivalent copy （GEC）/μL,no cross-reaction of othermicroorganisms,wide dynamic linear relationship in the template range of 4.62GECto 4.62×10⁹GEC （R²=0.997）,and high precision of the intra-and inter-assayvariations lower than 2% and 5%,respectively.To the detection of typical pathogenic bacteria,qualitative PCR method andreal-time fluorescent quantitative PCR of Salmonella typhi,Shigella,Escherichia coliand Vibrio cholerae were established,respectively.Using standard strains ofSalmonella typhi,Shigella and Escherichia coli,the standard curve of real-timequantitative PCR,the dynamic linear relationship in the template range of 0.366CFU～3.66×10⁵CFU,0.44CFU～4.4×10⁵CFU,and4.56CFU～4.56×10⁵CFU,respectively.Applying these PCR detection methods to surface waters （source of drinking water-Heihe River,landscape and recreational water-Xingqinghu Lake and BeihuLake,urban river-Chanhe River）and reference water-secondary effluent in Xi’ancity,the distribution and variation of typical pathogens in various environmentalwaters were researched by one year surveillance.It was found that enterovirusespresented in surface waters and secondary effluent,and the concentration obeyedlog-normal distribution.The average concentration of enteroviruses in Heihe River,Xingqinghu Lake,Beihu Lake,Chanhe River and secondary effluent was of 47GEC/L,127GEC/L,120GEC/L,422GEC/L and 746GEC/L,respectively.For HeiheRiver,most of enteroviruses positive samples and high concentration samplespresented in spring and summer.For Xingqinghu Lake and Beihu Lake,the period ofhigh concentration and high positive rate were in late of summer and early of autumn.Obvious fluctuation of enteroviruses concentration with change of season wasobserved in Chanhe River.For secondary effluent,early of September tomid-December and late of April to the end of May were two periods of highconcentration of enteroviruses.The fact that high similarity of nucleotide sequencesfrom various water samples indicated that the single species of enterovirusespresented in water environment.Salmonella typhi,Shigella,Escherichia coli,and Vibrio cholerae were detected by PCR method,and total bacteria,total coliform and fecal coliform were alsodetected by traditional culture method.The results showed that Vibrio cholerae wasnegative in all waters and the concentrations of other pathogenic bacteria were subjectto the lognormal distribution.The detection results of Salmonella typhi in samplesfrom Heihe River,Xingqinghu Lake and Beihu Lake were all negative.It was foundthat high concentration of Salmonella typhi and no obvious regularity of concentrationvariation in Chanhe River and secondary effluent.Shigellae were found in all waters.The concentration of Shigella was low before the summer,but it increased rapidly insummer.The concentration of Escherichia coli gradually rose from winter to summer.By analyzed the relationship between enteroviruses,enteric pathogenic bacteria,bacteriological index and physical-chemical index,the results indicated thatbacteriological index can not replace the detection of enteroviruses because of noevident correlation between them.In many waters,total bacteria count,total coliformand fecal coliform showed positive correlated with water temperature,turbidity andCOD,and negative correlated with DO.When the concentration of total coliform orfecal coliform was more than 10³CFU/100mL,Salmonella typhi increased rapidly. Shigella was significantly positive correlation with bacteriological index,and theconcentration gradually increased with concentration of coliforms or fecal coliforms,but Shigella presented in the waters with low concentration of coliform.Based on the PCR results of enteric pathogens,Human health risk associatedwith pathogens in various waters was evaluated combined with exposure assessmentand dose-response analysis."Enteric pathogens of maximum risk" in various waterswas determined and requirement of risk control was also proposed.The result showedthat enteroviruses were enteric pathogens of maximum risk in Heihe River.Enteroviruses must be effectively removed to ensure the safety of drinking water.When virus removal reached 6-log,the probability of enteroviruses infection riskbelow acceptable annual risk level (10^-4/a)was more than 90%.For Xingqinghu Lakeand Beihu Lake,enteroviruses were still major health risk source.When virusremoval was 4-log,the safety was more than 90%.For Chanhe River,major healthrisk was multiple.When direct as landscape and recreational water,health risk wasmainly caused by Shigella and Escherichia coli.Because of different variation ofenteroviruses and enteric pathogenic bacteria,water safety associated withenteroviruses was low when the removal of virus and pathogenic bacteria were 4-log.更多还原