采用套式PCR检测水库产毒微囊藻

根据所测定的微囊藻毒素合成酶mcyB基因的部分核苷酸序列,设计并筛选出两对特异性引物,用于产毒微囊藻的套式PCR检测。套式PCR针对毒素基因的检测结果与ELISA针对微囊藻毒素的检测结果相一致,但灵敏度更高。套式PCR的检测下限达1—10个微囊藻细胞/反应。采用套式PCR对广东12个主要供水水库的247份水样进行了产毒微囊藻检测,共检出阳性水样82份,阳性率为33.2%。这些阳性水样分布于除深圳水库以外的其他11个水库;其中汤溪水库水样套式PCR检出阳性率最高,达67.4%,其水样一步PCR的检出阳性率亦达25.6%,值得引起水文部门重视,并进行进一步跟踪监测。     

微囊藻毒素合成酶基因的PCR检测方法

针对微囊藻毒素合成酶基因簇的核酸序列,筛选特异性引物,探索一种适用于自然水样中微囊藻产毒潜能检测的全细胞PCR方法。经灵敏度测试表明,这种PCR方法的检测下限相当于100cells。该方法不需要提取基因组DNA,检测所需水样量少,具有操作简便、快速、成本低、灵敏度高等优点,能应用于水库等饮用水源水体中具有产毒潜能的微囊藻的检测。     

全细胞多重PCR检测蓝藻、微囊藻及产毒微囊藻方法初探

选取三对分别针对微囊藻、蓝藻16S rDNA及微囊藻毒素合成酶基因mcyB的保守序列的特异性引物209F/409R、27F1/409R、MTR/MTF,其中409R为一条共用引物。设计并优化了一种可以同时检测蓝藻和微囊藻的两重全细胞PCR方法和一种可以同时检测蓝藻、微囊藻和可产毒微囊藻的三重全细胞PCR方法,并且测试了这两种PCR反应的灵敏度区间,分别为10⁵～10³cell·mL^-1、10⁵～10²cell·mL^-1。对采集水库水样检测结果表明双重全细胞PCR方法可以直接应用于对天然水样的检测,三重全细胞PCR方法可用于实验室培养藻细胞的筛查。全细胞多重PCR方法具有快速、简便、准确等特点,在水体微囊藻毒素检测预警方面具有应用价值。     

应用双重PCR检测环境水体中的军团菌

建立双重PCR方法以检出环境水体中的军团菌。设计两对引物,分别扩增军团菌的16S rRNA和M ip基因,扩增片段长各为375bp和996bp。该方法检测军团菌的灵敏度为5.8×102cfu/m l,6株嗜肺标准军团菌均扩增出996bp和375bp两条带,4株非嗜肺军团菌扩增出375bp条带,4株非军团菌无条带;检测71份环境水样,5份出现两条条带,2份可见375bp条带,阳性率为7.0%。该方法快速、灵敏、特异,为水体中的嗜肺军团菌检测提供了有效方法。     

水华蓝藻产毒特性的PCR检测法

特异引物对（TOX 1P／1F;TOX 2P／2F）用于检测微囊灌毒素合成酶基因mcyB片段在38种水华蓝藻中的分布情况。结果显示,所有能产生微囊灌毒素的微囊藻都有特异扩增条带,非产毒株则没有,几种常规的毒性检测方法验证了PCR方法所获结果的准确性。本研究发展了以全细胞PCR法检测mcyB片断,说明全细胞PCR检测法适用于不同来源的蓝藻材料。结果证明以DNA为基因鉴别产毒和非产毒微囊藻及其他水华蓝藻的方法是可行的和实用的。     

利用3对引物鉴定产毒和非产毒微囊藻

根据微囊藻毒素合成酶基因簇序列 ,合成了 3对引物epF/mb1R ,mcF/teR ,mcF/umR ,通过全细胞PCR的方法检测了 19种不同来源微囊藻产毒的情况。 3种引物对 15株产毒微囊藻中均可扩增到预期大小的片段 ,测序结果证明这些片段是微囊藻毒素合成酶基因片段。PCR反应结果与HPLC分析所得到的结果有良好的对应性。在此基础上 ,初步确定了 3对引物检测产毒微囊藻对细胞浓度要求的下限。与其它引物相比 ,3对引物的特异性强 ,扩增条带大小适中 ,便于观察     

冬季太湖表层底泥产毒蓝藻群落结构和种群丰度

应用荧光定量PCR对冬季太湖不同湖区底泥表面有毒微囊藻和总微囊藻种群丰度进行调查,同时基于PCR-DGGE技术对底泥中有毒微囊藻群落结构进行分析。结果表明:微囊藻在太湖底泥表面分布广泛,所有采样点都检测到有毒微囊藻存在,且不同湖区有毒微囊藻和总微囊藻种群丰度存在显著差异,有毒微囊藻和微囊藻基因型丰度范围分别为1.23×10⁴-3.75×10⁶拷贝数/g干重和2.56×10⁴-1.07×10⁷ 拷贝数/g干重,有毒微囊藻与微囊种群丰度的比例为4.8％-35.2％;DGGE指纹图谱显示,冬季太湖不同湖区表层底泥中有毒微囊藻群落结构相似性较高,相似性系数为70.2％-96.0％。虽然不同湖区基因型组成存在差异,但所有样品中占优势的基因型是一致的。同时发现,优势基因型所占的比例与样品的香农多样性指数呈负相关。序列分析表明,mcyA序列长度为291bp,序列相似性超过97％。综合定量PCR结果和底泥中叶绿素a和藻蓝素浓度的测定结果,可以得出2010年冬季太湖蓝藻越冬主要集中在梅梁湾、竺山湾、贡湖湾和湖心。通过建立荧光定量PCR分析方法,为研究湖泊底泥中蓝藻种群丰度动态变化奠定了基础。     

阿氏浮丝藻mcyT基因序列多样性研究

为研究我国浮丝藻（Planktothrix Anagnostidis et Komrek）的毒素相关基因,选取分离自我国不同省份水体的13株阿氏浮丝藻,通过PCR检测其微囊藻毒素合成酶基因mcyA、mcyE及mcyT研究其毒素基因特性。PCR结果表明除mcyT之外其他引物检测均无扩增产物,说明这13株浮丝藻不具备产微囊藻毒素的能力。通过克隆测序得到mcyT序列,并进行分子系统分析,构建了关于mcyT序列的Neighbor-Joining系统树,结果表明mcyT序列可以将产毒与不产毒浮丝藻分为两大独立的分支,两个分支之间的最低序列相似度分别为98.5%和99.1%。研究结果可为后续研究我国浮丝藻的微囊藻毒素合成相关基因的多样性以及分子监测提供参考。       

饮用水处理流程中的微藻及其去除率

为了解某市饮用水处理流程中的藻类状况,对某市饮用水源及自来水厂各主要处理过程中的水样进行藻类检测鉴定。在各水样中共检测到藻类72种,蓝藻、绿藻和硅藻为优势藻群,并检出微囊藻、颤藻和鱼腥藻等潜在产毒的种类,其中微囊藻在源水中的细胞密度为1.9×106cell·L-1。源水中藻类细胞密度为2.8×106cell·L-1,处理后自来水中藻类的细胞密度为4.6×104cell·L-1,藻类总去除率为98.3%,其中滤池对藻类的去除率为82.7%,但其对蓝藻的去除率仅为11.8%。研究结果表明,该市水源可能已受到藻毒素的污染;现有自来水处理工艺中的滤池难以对个体较小的有毒蓝藻有效去除。针对该地区水源藻类的分布情况提出了优化过滤工艺的建议。     

荧光原位杂交技术和流式细胞仪用于环境样品中产毒及非产毒微囊藻的定量监测

全球范围内,高频次、大范围暴发的蓝藻水华对淡水水体环境造成严重影响.微囊藻因其在生长特别是衰亡过程中向水体释放微囊藻毒素而威胁人类健康.因此,分析其产毒株及非产毒株在环境样品中的组成,建立产毒蓝藻的预报及评价体系显得极为重要.本文采用荧光原位杂交技术结合流式细胞技术实现对环境样品中产毒藻株的鉴别与定量.针对目标基因mcyA设计的、以地高辛标记的双链DNA探针可有效应用于产毒微囊藻FACHB905和PCC7806的鉴别.分别对来自滇池、太湖和关桥的11个样品进行分析显示,该方法与传统的形态学鉴定及PCR方法有较好的匹配.荧光原位杂交技术与流式细胞相结合可有效鉴别产毒与非产毒微囊藻,尤其可以对野外样品中产毒与非产毒藻株进行简便、可视化地鉴别,从而达到对产毒微囊藻水华早期预警的目的.     

Overwintering of Microcystis in Lake Mendota

SUMMARY. Microcystis , an important bloom-forming blue-green alga in Lake Mendota, Wisconsin, was found to accumulate at the lake bottom and overwinter there. Microcystis collected from winter sediments were photosynthetically active, showing temperature and light optima similar to summer and autumn populations. Sediments incubated under in situ conditions in the laboratory maintained photosynthetic activity for at least four months. Such a maintenance ability may be part of an overall strategy by Microcystis populations to ensure a suitable inoculum for the following growth season.     

A PCR‐BASED TEST TO ASSESS THE POTENTIAL FOR MICROCYSTIN OCCURRENCE IN CHANNEL CATFISH PRODUCTION PONDS1,2

Microcystis aeruginosa is a common form of cyanobacteria (blue‐green algae) capable of forming toxic heptapeptides (microcystins) that can cause illness or death. Occasionally, blooms of cyanobacteria have caused toxic fish‐kills in catfish production ponds. We have developed a PCR test that will detect the presence of microcystin‐producing cyanobacteria. Microcystin producers are detected by the presence of the microcystin peptide synthetase B gene (an obligate enzyme in the microcystin pathway), which appears to be present only in toxin‐producing cyanobacteria. These PCR amplifications can be performed in multiplex using purified DNA from pond waters or by two‐stage amplification from native water samples. A synoptic survey of 476 channel catfish production ponds from four states in the southeastern United States revealed that 31% of the ponds have the genetic potential to produce microcystins by toxic algae.     

七株微囊藻系统进化关系的RAPD-PCR分析

应用RAPD-PCR的方法,选用24个随机引物,分析来自不同地区的7株微囊藻的基因组多态性。结果显示,Microcystis.viridis及M.wesenbergii明显与M.aeruginosa区分开。M.aeruginosa分为两个可视为不同种的异源分类单位。作为对照的Anabaena sp.7120与其他微囊藻株表现出完全不同的基因型及更远的遗传距离。 此项研究表明,以基因型而不是表现型为基础,分析蓝藻种内及种间区别是可能的。因此,为解决蓝藻分类问题,特别是在种和属的水平上,提供了重要的线索。结合正在进行的用特异性及准确性强的引物区分微囊藻产毒及非产毒株的方法,RAPD-PCR可望将微囊藻产毒及非产毒株进化关系澄清。     

Cellulolytic decomposition in streams of contrasting pH and its relationship with invertebrate community structure

SUMMARY. 1. Ceilulolytic decomposition, measured by loss of tensile strength in strips of cellulose test cloth, was estimated in thirty-four stream sites in experiments in summer, autumn and winter.
2. The results of multiple regression analyses showed that strength loss was most closely related to temperature in summer, when the model accounted for only 38% of the variation, and to stream pH in autumn and winter, when the models accounted for 52% and 75% of the variation, respectively.
3. The relationship between decomposition rate and the structure of invertebrate communities in streams of dissimilar pH is discussed. We argue that a very rapid rate of decomposition of coarse particulate organic matter may profit consumers of fine particulate organic matter at the expense of coarse particle shredders.     

Competition for space by predators in streams: field experiments on a net-spinning caddisfly

SUMMARY. 1. Field experiments in a fishless stream were carried out on an abundant caddisfly with a predatory, net-spinning larva, Plectrocnemia conspersa (Curtis), to assess whether net site availability affects their microdistribution.
2. Net sites were supplemented by adding nought, one or four artificial structures to replicated patches on the stream bed. In each of three experiments at different seasons (summer, autumn and late winter), caddis densities increased significantly in patches with extra net sites.
3. The response of caddis to supplemented net sites could be affected by the subsidiary effects of food and offish. These potential interactions were assessed in each experiment by varying net site density in two additional treatment stretches in which (1) prey abundance was increased by releasing Daphnia , and (2) brown trout ( Salmo trutta L.) were enclosed. The responses of caddis in these two treatments were compared to that in the reference stretch, where only net site density varied.
4. Increased food abundance enhanced the response of caddis to net site supplementation in winter, when natural prey was least abundant, but not in summer or autumn. We suggest that extra food affects the mechanism determining net building only when prey availability is below some threshold.
5. The presence of fish precluded any effect of extra net sites in summer, but had no effect in autumn (the winter fish treatment was lost). We suggest fish predation reduced the densities of caddis in summer, so that net sites no longer limited local densities. In autumn, fallen leaves provided refugia from fish, which consequently were less effective predators of P. conspersa.     

Monitoring changing toxigenicity of a cyanobacterial bloom by molecular methods

Cyanobacterial blooms are potential health hazards in water supply reservoirs. This paper reports analyses of a cyanobacterial bloom by use of PCR-based methods for direct detection and identification of strains present and determination of their toxigenicity. Serial samples from Malpas Dam, in the New England region of Australia, were analyzed during a prolonged, mixed cyanobacterial bloom in the summer of 2000 to 2001. Malpas Dam has been shown in the past to have toxic blooms of Microcystis aeruginosa that have caused liver damage in the human population drinking from this water supply reservoir. Cyanobacterial genera were detected at low cell numbers by PCR amplification of the phycocyanin intergenic spacer region between the genes for the beta and alpha subunits. The potential for microcystin production was determined by PCR amplification of a gene in the microcystin biosynthesis pathway. The potential for saxitoxin production was determined by PCR amplification of a region of the 16S rRNA gene of Anabaena circinalis strains. Toxicity of samples was established by mouse bioassay and high-pressure liquid chromatography. We show that bloom components can be identified and monitored for toxigenicity by PCR more effectively than by other methods such as microscopy and mouse bioassay. We also show that toxigenic strains of Anabaena and Microcystis spp. occur at this site and that, over the course of the bloom, the cell types and toxicity changed. This work demonstrates that PCR detection of potential toxicity can enhance the management of a significant public health hazard.     

Correlation between the morpho-cytohistochemistry of the outer mantle epithelium of Anodonta cygnea with seasonal variations and following pollutant exposure

Seasonal and experimental conditions induce morphological and cytochemical variations in the outer mantle epithelium (OME) of the freshwater bivalve Anodonta cygnea, probably influencing the shell calcification mechanism. In this study, OME samples were taken from untreated animals in autumn, winter, spring and summer as well as from animals exposed to divalent metals (cadmium, chromium, lead, copper and zinc) and pesticides (setoxidim and dimethoate) and observed by light microscopy. The present results showed that OME cells have larger cell volumes and increased amounts of secreted macromolecules during spring and summer than in autumn and winter. This correlates with higher shell calcification rates in spring and summer and lower shell calcification rates in autumn and winter. The experiments showed that incubation with pollutants for 8 months dramatically reduced the cellular volume so that the density of cytoplasmic material appeared higher that in the control samples. The pronounced changes in OME cells suggest a significant decrease in secretory activity following exposure to toxic agents and this has implications for the shell calcification process.