淡水水体中微囊藻毒素的毒性鉴别和测定技术

微囊藻毒素是一类环状多肽类物质,具有很强的肝毒性.从化学分析、生物检测以及生物化学分析三个方面,对目前世界范围内有关微囊藻毒素鉴定和分析的测试技术进行了综述,指出了各类技术的优缺点,同时给出了各种测定方法对于不同介质样品的检出限.     

Detection of Human Enteric Viruses in Freshwater from European Countries

The transmission of water-borne pathogens typically occurs by a faecal–oral route, through inhalation of aerosols, or by direct or indirect contact with contaminated water. Previous molecular-based studies have identified viral particles of zoonotic and human nature in surface waters. Contaminated water can lead to human health issues, and the development of rapid methods for the detection of pathogenic microorganisms is a valuable tool for the prevention of their spread. The aims of this work were to determine the presence and identity of representative human pathogenic enteric viruses in water samples from six European countries by quantitative polymerase chain reaction (q-PCR) and to develop two quantitative PCR methods for Adenovirus 41 and Mammalian Orthoreoviruses. A 2-year survey showed that Norovirus, Mammalian Orthoreovirus and Adenoviruses were the most frequently identified enteric viruses in the sampled surface waters. Although it was not possible to establish viability and infectivity of the viruses considered, the detectable presence of pathogenic viruses may represent a potential risk for human health. The methodology developed may aid in rapid detection of these pathogens for monitoring quality of surface waters.     

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。     

免疫磁珠分离与实时定量PCR技术联合检测水中轮状病毒的研究

建立了一种免疫磁珠分离技术联合实时定量PCR快速定量检测水中轮状病毒的方法.通过制备能够分离水中轮状病毒的特异免疫磁珠,优化分离条件,建立了免疫磁珠分离前处理方法,并与逆转录、实时定量PCR结合,成功用于水中轮状病毒的检测.研究发现,在1 mL水样中加入10 μL轮状病毒免疫磁珠、0.25 μL Tween 20、孵育2 h可达到较好的分离效果;免疫磁珠可用于3%牛肉浸膏等常用病毒洗脱液中的轮状病毒的分离,表明该分离技术能与已有的病毒浓集方法良好地整合.免疫磁珠分离技术与实时定量PCR联合用于检测水中轮状病毒,全过程需时约5 h,检测限为1×10⁴　copies/mL(相当于3～4　PFU/mＬ),检测结果与细胞病变试验检测结果有良好的线性相关关系（R²=0.981 6）,表明其能较好地表征水样的病毒感染风险.对接种已知量的轮状病毒的污水处理厂二级出水、再生水、地表水、自来水等水样的实验表明,该法可用于各种水样中轮状病毒的检测.     

Detection,Quantification, and Microbial Risk Assessment of Group A Rotavirus in Rivers from Uruguay

Food and Environmental Virology - The aim of this study was to detect, quantify, and assess the risk of infection and illness for Group A Rotavirus (RVA) in the watersheds of the Santa Lucia and...     

水中轮状病毒实时定量PCR外标准品的构建

采用细胞培养和T-A克隆技术,在轮状病毒主要结构蛋白VP7基因序列上设计合成引物,经PCR扩增后将特异性产物连接入pGEM-T-easy载体中,经过酶切鉴定和测序分析获得轮状病毒cDNA标准品.利用常规PCR和实时定量PCR方法对所获得的cDNA标准品进行特异性、稳定性和重复性指标的检验.结果表明,利用此标准品制备的标准曲线具有较高的扩增效率和良好的线性关系(斜率为-3.353,R2=0.995);实时定量PCR熔解曲线分析表明,温度在81℃±0.5℃的PCR产物是轮状病毒VP7序列上的特异性产物,表明此标准品是轮状病毒特异性的;同时,所制备的标准品在实时定量PCR检测中具有较大的线性范围(9×100～9×1011 copies/μL,每个反应最低可以检测到9个拷贝数的轮状病毒cDNA,表明利用该标准品进行实时定量PCR分析时具有较高的检测灵敏性;此外,该标准品具有较高的稳定性和重复性(3次独立实验CV值在0.2%～0.9%之间),可长期稳定保存.构建的标准品可用作检测水环境中轮状病毒的实时荧光定量PCR的外标准品.     

Detection of Norovirus Recombinant GII.2[P16] Strains in Oysters in Thailand

Human norovirus causes sporadic and epidemic acute gastroenteritis worldwide, and the predominant strains are genotype GII.4 variants. Recently, a novel GII.17[P17] and a recombinant GII.2[P16] strain have been reported as the causes of gastroenteritis outbreaks. Outbreaks of norovirus are frequently associated with foodborne illness. In this study, each of 75 oyster samples processed by a proteinase K extraction method and an adsorption-elution method were examined for noroviruses using RT-nested PCR with capsid primers. Thirteen (17.3%) samples processed by either method tested positive for norovirus genogroup II (GII). PCR amplicons were characterized by DNA sequencing and phylogenetic analysis as GII.2 (n?=?6), GII.4 (n?=?1), GII.17 (n?=?3), and GII.unclassified (n?=?3). Norovirus-positive samples were further amplified by semi-nested RT-PCR targeting the polymerase-capsid genes. One nucleotide sequence revealed GII.17[P17] Kawasaki strain. Five nucleotide sequences were identified as belonging to the recombinant GII.2[P16] strains by recombination analysis. The collected oyster samples were quantified for norovirus GII genome copy number by RT-quantitative PCR. Using the proteinase K method, GII was found in 13/75 (17.3%) of samples with a range of 8.83–1.85?×?10⁴ genome copies/g of oyster. One sample (1/75, 1.3%) processed by the adsorption-elution method was positive for GII at 5.00?×?10¹ genome copies/g. These findings indicate the circulation of a new variant GII.17 Kawasaki strain and the recombinant GII.2[P16] in oyster samples corresponding to the circulating strains reported at a global scale during the same period of time. The detection of the recombinant strains in oysters emphasizes the need for continuing systematic surveillance for control and prevention of norovirus gastroenteritis.

     

Contamination of Clams with Human Norovirus and a Novel Hepatitis A Virus in Cameroon

Food and Environmental Virology - Shellfish constitute an important protein source but may be contaminated by viruses from various origins. A study performed on clams collected in Cameroon showed a...     

Application of a Swab Sampling Method for the Detection of Norovirus and Rotavirus on Artificially Contaminated Food and Environmental Surfaces

Noroviruses and rotaviruses are the leading causes of non-bacterial gastroenteritis in humans worldwide. Virus-contaminated food and surfaces represent an important risk to public health. However, established detection methods for the viruses in food products are laborious and time-consuming. Here, we describe a detailed swabbing protocol combined with real-time RT-PCR for norovirus and rotavirus detection on artificially contaminated food and environmental surfaces. Recovery rates between 2 and 78% for norovirus and between 8 and 42% for rotavirus were determined for contaminated food surfaces of apple, pepper, cooked ham and salami. From contaminated environmental surfaces (stainless steel, ceramic plate, polyethylene, wood), recovery rates between 26 and 52% (norovirus) and between 10 and 58% (rotavirus) were determined. The results demonstrate the suitability of the swab sample method for virus detection on food and environmental surfaces. Compared to other methods, it is easy to perform and significantly time-saving, predestining it for routine testing.     

中国的淡水资源问题

我国单位面积淡水资源接近全球平均值，但由于人口密度比世界平均大3倍，人均淡水资源仅为世界人均值的1/3弱。淡水是可再生资源，主要靠自然降水补给。地表水体传导水的能力强，但存储容积小；地下水含水层传导水的能力弱，但存储容积大，其静态库容只是调节空间；深层封闭地下水资源潜力不大，长期大量开发会导致地面沉降等严重后果。我国大气降水极不均匀的时空分布，使蒸发量和流失量加大，农业灌溉更加剧了淡水资源的供需矛盾。联合调度地表水和地下水，可以实现淡水资源在时间上的调控。淡水又是无法依赖进口的资源，只能依靠节水和现有淡水资源的科学使用。我国淡水资源南多北少，应当避免人口过多地向缺水地区流动。南水北调可以缓解北方缺水的局面，但应主要用于特殖干旱年份，供水的基本来源仍应立足本地。我国南方缺水主要是污染造成的，应当集中力量治理污染。今后，有必要加强流域的统一管理，规定第一地区的取水量和排污总量的限额。     

Detection of Human Adenovirus,Rotavirus, and Enterovirus in Tap Water and Their Association with the Overall Quality of Water in Karachi,Pakistan

Food and Environmental Virology - Drinking water supplies in the developing world often serve as a biosphere for various organisms. Viral gastroenteritis is a neglected area of research in...     

Accumulation and Depuration Kinetics of Rotavirus in Mussels Experimentally Contaminated

Bivalve mollusks as filter-feeders concentrate in their digestive tissue microorganisms likely present in the harvesting water, thus becoming risky food especially if consumed raw or poorly cooked. To eliminate bacteria and viruses eventually accumulated, they must undergo a depuration process which efficacy on viruses is on debate. To better clarify the worth of the depuration process on virus elimination from mussels, in this study we investigated rotavirus kinetics of accumulation and depuration in Mytilus galloprovincialis experimentally contaminated. Depuration process was monitored for 9 days and virus residual presence and infectivity were evaluated by real time quantitative polymerase chain reaction, cell culture and electron microscopy at days 1, 2, 3, 5, 7, 9 of depuration. Variables like presence of ozone and of microalgae feeding were also analyzed as possible depuration enhancers. Results showed a two-phase virus removal kinetic with a high decrease in the first 24 h of depuration and 5 days necessary to completely remove rotavirus.     

Quantitative Detection of Human Adenovirus and Human Rotavirus Group A in Wastewater and El-Rahawy Drainage Canal Influencing River Nile in the North of Giza,Egypt

Food and Environmental Virology - Environmental monitoring is critical in a developing country like Egypt where there is an insufficient framework for recording and tracking outbreaks. In this...     

淡水沉积物中重金属生物有效性的研究进展

淡水重金属污染已成为全球关注的热点环境问题之一。沉积物作为重金属的重要储库与污染内源,可对水环境质量及水生态系统产生严重危害。生物有效性是评价重金属生物可利用性及其毒性效应的关键指标,研究淡水沉积物中重金属的生物有效性与调控机制,有助于阐明重金属的污染机理及其生态环境效应,可为重金属的污染防治提供科技支撑。本文回溯了沉积物重金属生物有效性的定义内涵、发展历史,重点介绍了重金属生物有效性评价的主要方法、适用性及其优缺点,归纳了影响重金属生物有效性的主要因素与内在机制,概述了目前国内外典型河流、湖泊和水库等淡水水体沉积物中相关研究的最新进展。本文进一步指出,在今后研究中亟需整合现有分析方法,形成统一的沉积物重金属生物有效性评价指标与方法体系,并开展权威实验室比对研究。同时,应考虑不同淡水生态系统的区域差异性,采用生物监测方法与原位被动采样技术相结合,将生物配位体模型纳入重金属的生态毒理效应研究中。最终,依据不同淡水环境的特性,尽快建立相应的沉积物重金属质量基准,加强重金属环境污染的科学管理。     

Rotavirus Occurrence in Shellfish with Low Levels of E. coli

The purpose of this study was to evaluate “in field” the accumulation of virus in shellfish and compare it with the concentration of bacterial indicators. Individuals of Mytilus galloprovincialis were placed in two sampling station located in a contaminated coastal bay and in one control station located one kilometer offshore. The presence of Rotavirus and E. coli was assessed weekly both in seawater and in shellfish samples. The Rotavirus genome was detected in water, preliminarily concentrated by tangential flow ultrafiltration method, and in hepatopancreas of mussels by Real-Time PCR. E. coli was enumerated in water matrices by a filtering method and in mussels by the MPN method. Rotaviruses were not recorded in seawater, while in mussels they were detected since third week after placement. E. coli in mussels were always below the limits set in the Regulation (EC) 854/2004. This study suggests the need for a viral indicator to insure the safety for consumption of shellfish.     

文蛤和四角蛤蜊对石油烃富集排除及损伤效应

海水石油烃含量的增加对贝类等水产动物生长发育造成了严重威胁,研究文蛤和四角蛤蜊在石油烃影响下组织富集排除规律及氧化损伤效应是非常必要的。该研究将文蛤和四角蛤蜊在石油烃浓度为0.1、0.3、1.0和3.0 mg/L海水中分别暴露15 d,然后在清洁海水中排除15 d,测定消化盲囊和鳃丝石油烃蓄积量、生物浓缩系数和丙二醛含量。结果显示,文蛤和四角蛤蜊在暴露期间各组织石油烃含量随时间不断升高,且石油烃浓度越高,蓄积量越大,生物浓缩系数越小;0.1 mg/L和0.3 mg/L石油烃暴露组的文蛤能将石油烃完全排除,四角蛤蜊只能将0.1 mg/L暴露组蓄积的石油烃完全排除,1.0 mg/L及以上暴露组2种蛤均不能完全排除体内的石油烃;2种蛤丙二醛含量在暴露期间均不断上升,且0.1 mg/L暴露组通过排除阶段能恢复到正常水平,而0.3 mg/L以上暴露组只有文蛤消化盲囊丙二醛含量能回到对照组水平,其它处理组丙二醛含量在排除阶段后期仍然高于对照组。结果证实,文蛤和四角蛤蜊对石油烃的蓄积能力较强,对0.3 mg/L及以下浓度石油烃具有一定耐受性,而1.0 mg/L浓度以上石油烃导致两种蛤氧化损伤较明显,四角蛤蜊各指标变化较文蛤显著,四角蛤蜊作为石油烃监测种较文蛤更敏感,且同种指标鳃丝较消化盲囊受影响更显著,2种蛤的蓄积量和丙二醛含量存在一定的正相关关系,各指标均能反映不同浓度石油烃对文蛤和四角蛤蜊影响规律,可以作为监测海洋石油烃污染的生物学指标。     

流域土地利用变化的水生态响应研究

流域土地利用变化造成水体物理、化学条件变化,并对诸多水生生物产生影响,进而破坏水系的生态系统结构和功能.研究显示:① 流域土地利用变化将引起水文、底质、生境改变等物理响应,以及水体营养盐、细菌、有毒有害物含量、溶解氧变化等化学响应,分布式水文模型成为研究物理和化学响应的重要工具;② 流域土地利用变化的生物响应表现为水生生物与自然用地类型呈正相关,而与农业用地、城镇用地呈负相关,并且存在明显的尺度效应,统计学分析是主要的研究方法;③ 大尺度的土地利用变化在水文过程下造成河段尺度上的外源物质输入、水文条件、底质结构等物理化学因子变化,是影响水生生物生境的直接原因.存在主要问题:① 流域土地利用变化的生物响应关系机制尚不明晰;② 流域土地利用变化的生物响应是通过物理响应、化学响应来间接作用的,但三者之间的非线性关系十分复杂;③ 水生生物对环境因子变化的敏感性和适用性存在较大差异.因此,未来研究应关注流域水文过程驱动下的物质迁移转化过程及由此带来的物理、化学和生物响应以及三者之间的关系,加强流域水文规律总结和参数率定,构建符合流域特征的参数数据库,并着力构建以保护水生态系统结构与功能为核心的流域山水林田湖系统综合管控模式.