逆转录-聚合酶链反应(RT-PCR)技术同时检测水中多种肠道病毒

建立了一种利用通用引物RT-PCR技术检测水中肠道病毒的方法.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,根据肠道病毒RNA5′非编码区中具有高度同源性的序列来设计通用引物.比较了M-MLV酶和AMV酶的逆转录效果,AMV酶能够成功地从地表水和生活污水中逆转录病毒RNA,更适于实际应用.对比研究了PCR过程中的退火温度,c(Mg2＋)等因素对RT-PCR检测结果的影响,选择退火温度55　℃,c(Mg2＋)为2　mmol/L的反应条件,优化了RT-PCR检测方法.通过检测水样中接种的连续稀释的病毒,确定了该检测方法的灵敏度为38　CCID₅₀.考察人工污染的地表水、污水、二级处理出水样品发现,检测灵敏度基本一致.该方法可应用在实际环境的肠道病毒检测中.      

城市污水二级处理出水中沙门氏菌的PCR检测

建立了一种利用PCR检测城市污水二级处理出水中沙门氏菌的方法.采用膜吸附-洗脱法浓缩二级出水水样中的沙门氏菌,通过选用一对涵盖性和特异性均较强的引物139和141,对浓缩后的沙门氏菌进行PCR检测.利用正交试验法,对沙门氏菌PCR体系进行五因素四水平的试验,建立了沙门氏菌PCR的最佳体系.通过梯度PCR考察了退火温度对检测结果的影响,选择65℃作为退火温度,优化后沙门氏菌PCR体系的检测灵敏度可达0.314ngL.通过对已知细菌浓度的水样进行检测,确定了该法检测水样中沙门氏菌的灵敏度为2130CFUL.与传统MPN法的相比,该法在检测效果和检测效率上都优于MPN法.     

PCR技术检测水中轮状病毒的研究进展

聚合酶链式反应,即PCR(polymerase chain reaction)技术,是一种快速扩增DNA或cDNA序列的方法.水环境中的轮状病毒是导致世界范围内婴幼儿腹泻的主要原因,严重危害着水质安全和人类健康.建立和完善水中轮状病毒灵敏、快速的检测方法对预防和控制水体疾病的暴发具有重要的意义.随着分子生物学技术的发展,PCR及其衍生技术因其具有高度灵敏性、特异性和准确性等特点,成为检测水中轮状病毒最常用的方法.本文综述了利用RCR及其衍生技术,包括逆转录聚合酶链式反应、逆转录半巢式和巢式聚合酶链式反应、多重逆转录聚合酶链式反应、免疫聚合酶链式反应、与免疫磁珠分离相结合的聚合酶链式反应、与细胞培养相结合的聚合酶链式反应及实时定量聚合酶链式反应等,检测水环境中轮状病毒的研究进展,并分析了这些PCR技术在检测水环境中轮状病毒时存在的问题及其应用前景.     

生物填料反应器在二级处理出水溶度处理中的应用研究

研究了生物填料反应器（简称ＰＢＲ）用于二级处理出水深度处理时，对ＣＯＤ，ＳＳ，ＮＨ３－Ｎ的去除效果及影响因素。并推荐废水再用流程为：二级处理出水→上向流ＰＢＲ→双层滤料滤池→出水再用。推荐流程可有效地去除二级处理出水中的ＣＯＤ，ＳＳ，ＮＨ３－Ｎ，去除率分别为４２．５％，９２．３％．５２．６％。推荐流程出水可再用于生产，生活杂用，循环冷却水补充等用水场合。     

城市污水中人类星状病毒的检测及变化规律

人类星状病毒是典型的水媒病毒,可引起腹泻等疾病. 为了明确污水中人类星状病毒的分布及去除规律,利用常规PCR和定量PCR方法对西安市3个污水处理厂的进水和出水进行了为期1 a的监测;采用特异性引物,确立了人类星状病毒在环境水体中的定性PCR和荧光定量PCR的检测方法. 结果表明,人类星状病毒在进水和未消毒二级出水中呈较高的阳性率,分别为91.5%和36.6%. 经过核苷酸测序鉴定,人类星状病毒的主要血清型为HAst V-1和HAst V-4. 人类星状病毒在进水和未消毒二级出水中的平均浓度分别为1.47×104和1.03×103 copies/mL,污水处理厂对人类星状病毒的去除率为93.15%. 污水处理厂进水中人类星状病毒浓度呈季节性变化,春季和冬季较高,夏季和秋季较低,这一分布规律与该地区历年人类星状病毒的流行规律相似.      

自然光和UV辐照下二级出水DOM及毒性的变化

为了探明自然光和紫外光（UV）对二级处理出水中溶解性有机物（DOM）及其毒性效应的削减特性，研究了在自然光和UV两种不同光源的辐照条件下，二级处理出水中DOM的光谱特性、遗传毒性和植物毒性变化，阐明了毒性效应变化的成因.结果表明：相比于自然光照，UV辐照可以明显降低二级处理出水的UV₂₅₄，并达到21%矿化.自然光和UV均可降低二级处理出水中有色溶解性有机物（CDOM），但去除的组分有所差异.同时，自然光照与UV辐照均可降低二级处理出水的遗传毒性，其削减率分别达到92%和61%.遗传毒性的变化与CDOM荧光强度变化有显著相关性.UV辐照可降低二级处理出水的植物毒性，削减率达到81%，而自然光照对二级处理出水植物毒性的削减率仅为19%，削减效果较差.利用UPLC-MS检测二级出水中的除草剂，发现植物毒性削减的差异主要是由于自然光照下二级处理出水中的阿特拉津降解缓慢，表观降解速率常数为0.0033h^-1；但是UV辐照下二级处理出水中的阿特拉津可快速降解，其表观速率常数为0.2225h^-1.这为污水厂排放水毒性效应控制及生态安全保障提供了一定的依据.     

环境水体中肠道病毒的膜吸附-洗脱浓缩方法研究

在膜吸附-洗脱和洗脱液浓缩相结合的基础上,建立了一种简便实用的水中肠道病毒浓缩方法.通过实时定量RT-PCR检测,比较了不同材料和不同孔径的微孔滤膜对病毒的吸附效果;对膜洗脱方式进行了改进;研究了在洗脱液浓缩过程中,PEG浓度对于病毒回收率的影响.最后确定了最佳的浓缩方法.选择效果好而且来源广泛的0.22 μm孔径的混合纤维素酯微孔滤膜,采用磁力搅拌来洗脱滤膜上吸附的病毒;洗脱液浓缩步骤中,PEG最佳质量浓度为130　g/L.系统比较了不同病毒接种量下,方法中各步骤的病毒回收率.对接种已知量的肠道病毒的生活污水、二级处理出水和地表水等样品的试验结果表明,该方法效果稳定,适合不同水样中肠道病毒的浓缩分离.     

城市污水地下回灌深度处理技术

以城市污水地下回灌为回用目的,研究了不同的污水深度处理工艺,并着重研究了去除二级生物处理出水中有机物的处理工艺.研究结果表明,采用臭氧氧化和粉末活性炭预处理后再经土壤渗滤回灌,可大大改善水质,水中溶解性有机碳浓度可降至3mg／L以下,通过静态吸附实验选出了适于处理二级出水的粒状活性炭,并运用ADSA软件对有机物在活性炭上的吸附行为进行分析.将活性炭的动态实验同混凝、过滤结合起来,研究不同组合工艺对有机物的去除效果,筛选出了适于城市污水处理厂二级出水地下回灌的预处理工艺,其出水溶解性有机碳浓度低达3～4．5mg／L以下.     

湖泊中军团菌的巢式PCR-MPN检测法

为了直接检测环境水体中军团菌的存在,文章将巢式PCR与倍比稀释法相结合(巢式PCR-MPN法),采用两对引物扩增环境样品基因组DNA。将检测到含有军团菌的样品DNA模板进行十倍梯度稀释,由最低检测的稀释度,计算出样品中军团菌的细胞数,同时确定方法检测的灵敏度。并用克隆建库、构建系统树的方法来验证该文研究方法的特异性。研究结果表明,巢式PCR第二步后获得的序列均为军团菌序列,该方法军团菌DNA的检出限可以低至2.74 fg/μL。该研究建立的水体军团菌检测方法具有简便快捷、特异性好、灵敏度高的特点。     

生物填料反应器在二级处理出水深度处理中的应用研究

研究了生物填料反应器(简称PBR)用于二级处理出水深度处理时,对COD、SS、NP(?)-N的去除效果及影响因素。并推荐废水再用流程为:二级处理出水→上向流PBR→双层滤料滤池→出水再用。推荐流程可有效地去除二级处理出水中的COD、SS、NH_3-N。去除率分别为42.5％、92.3％、52.6％。推荐流程出水可再用于生产、生活杂用、循环冷却水补充等用水场合。     

Estimation of Contamination Sources of Human Enteroviruses in a Wastewater Treatment and Reclamation System by PCR–DGGE

A polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE) method was employed to estimate the contamination sources of human enteroviruses and understand how their dominant strains vary in a wastewater treatment and reclamation system consisting of sewage collection, wastewater treatment with membrane bioreactor and open lakes for reclaimed water storage and reuse. After PCR–DGGE using a selected primer set targeting enteroviruses, phylogenetic analysis of acquired enterovirus gene sequences was performed. Enteroviruses identified from the septic tank were much more diverse than those from grey water and kitchen wastewater. Several unique types of enterovirus different from those in wastewater samples were dominant in a biological wastewater treatment unit. Membrane filtration followed by chlorination was proved effective for physically eliminating enteroviruses; however, secondary contamination likely occurred as the reclaimed water was stored in artificial lakes. Enterovirus 71 (EV71), a hand-foot-and-mouth disease (HFMD) viral pathogen, was detected mainly from the artificial lakes, implying that wastewater effluent was not the contamination source of EV71 and that there were unidentified non-point sources of the contamination with the HFMD viral pathogen in the reclaimed water stored in the artificial lakes. The PCR–DGGE targeting enteroviruses provided robust evidence about viral contamination sources in the wastewater treatment and reclamation system.     

城市污水处理工艺对噬菌体的去除效果

测定了北京市3座城市污水处理厂各处理单元出水中的SC噬菌体和F-RNA噬菌体浓度进水中SC噬菌体和F-RNA噬菌体浓度范围分别为6.25 ×103～1.34×104 PFU·mL-1和2.4×10～2.4×103 PFU·mL-1,经过处理,上述2类噬菌体的总去除率分别为72.45%～99.89%和57.84%～93.06%,低于对粪大肠菌的处理效果生物曝气工艺对噬菌体的去除效率最高,深度处理中的砂滤工艺对噬菌体的去除作用不明显.根据测定的F-RNA噬菌体浓度预测水中肠道病毒的浓度,结果显示生活污水经常规处理和深度处理后仍含有0.65～15.8 PFU·L-1的肠道病毒.     

Large-Scale Survey of Human Enteroviruses in Wastewater Treatment Plants of a Metropolitan Area of Southern Italy

Human enteroviruses (HEVs) occur in high concentrations in wastewater and can contaminate receiving environmental waters, constituting a major cause of acute waterborne disease worldwide. In this study, we investigated the relative abundance, occurrence, and seasonal distribution of polio and other enteroviruses at three wastewater treatment plants (WWTPs) in Naples, Southern Italy, from January 2010 to December 2014. Influent and effluent samples from the three WWTPs were collected monthly. One hundred and sixty-one of the 731 wastewater samples collected (22.0%) before and after water treatment were CPE positive on RD cells; while no samples were positive on L20B cells from any WWTPs. Among the 140 non-polio enterovirus isolated from inlet sewage, 69.3% were Coxsackieviruses type B and 30.7% were Echoviruses. Among these, CVB3 and CVB5 were most prevalent, followed by CVB4 and Echo6. The twenty-one samples tested after treatment contained 6 CVB4, 5 CVB3, 3 Echo11, and 2 Echo6; while other serotypes were isolated less frequently. Data on viral detection in treated effluents of WWTPs confirmed the potential environmental contamination by HEVs and could be useful to establish standards for policies on wastewater management.     

河岸渗滤对不同工艺再生水补给景观水的处理功效

分别采用"二级出水+O3+GFH"和"二级出水+DNBF"两种不同工艺再生水补给人工湖,河岸渗滤(简称BF)作为净化再生水补给人工湖后的维护工艺,分析BF对人工湖中污染物的去除机制。结果表明:以"二级出水+O3+GFH"工艺出水作为人工湖补水,BF对NH4+-N、NO2--N、DOC的去除效率分别为85.1%、52.0%、22.5%。以"二级出水+DNBF"工艺出水作为人工湖补水,BF对NH4+-N、NO2--N、DOC的去除效率分别为64.4%、62.0%、26.4%。人工湖NH4+-N越高,水体中充分富氧,BF砂坝内硝化作用越强,BF对NH4+-N的去除率越高,可有效抑制蓝藻的暴发。     

Persistence of Viruses by qPCR Downstream of Three Effluent-Dominated Rivers in the Western United States

This study was designed to determine the quantitative polymerase chain reaction (qPCR) signal persistence of viruses in three effluent-dominated streams. Samples were collected from the effluent outfall of three wastewater treatment plants in the Western United States and downstream at different locations. All samples were tested for the presence of pepper mild mottle virus (PMMoV), adenoviruses, norovirus GI and GII, Aichi virus, and enteroviruses using qPCR. PMMoV was detected most frequently in 54/57 (94.7%) samples, followed by adenoviruses which was detected in 21/57 (36.8%) samples. PMMoV was detected at all locations downstream and up to 32 km from the discharge point. This study demonstrated that the detection signal of PMMoV was able to persist in wastewater discharges to a greater degree than human enteric viruses in effluent-dominated rivers.     

Transformation of erythromycin during secondary effluent soil aquifer recharging: Removal contribution and degradation path

Erythromycin (ERY), a widely used antibiotic, has recently been detected in municipal secondary effluents and poses serious threats to human health during wastewater reusing. In this study, the removal, fate, and degradation pathway of ERY in secondary effluent during soil aquifer treatment was evaluated via laboratory-scale SAT tests. Up to a 92.9% reduction of ERY in synthetic secondary effluent was observed in 1.0 m depth column system, which decreased to 64.7% when recharged with wastewater treatment plant secondary effluent. XRD-fractionation results demonstrated that the transphilic acid and hydrophobic acid fractions in secondary effluent compete for the adsorption sites of the packed soil and lead to a declined ERY removal. Moreover, aerobic biodegradation was the predominant role for ERY removal, contributing more than 60% reduction of ERY when recharged with synthetic secondary effluent. Destruction of 14-member macrocyclic lactone ring and breakdown of two cyclic sugars (l-cladinose and d-desosamine) were main removal pathways for ERY degradation, and produced six new intermediates.     

中水回用工程技术——膜生物反应器

抚顺市肉联厂的中水回用工程每日处理中水100m^3，采用MBR工艺。经过试运行后，CODCr去除率为51％，BOD5去除率为77％，悬浮物去除率为93％。实践证明，MBR工艺是一种简易、高效的中水回用技术。