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。     

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。     

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     

蓝藻毒素的类型及其产毒基因

水体富营养化及蓝藻水华的频繁发生,严重破坏水域生态系统,有毒水华蓝藻产生的藻毒素,污染水体且危害水生动物和人类健康。本文综述了水华蓝藻的次级代谢产物藻毒素及藻毒素合成基因的国内外研究进展,介绍了蓝藻毒素的类型、不同毒素毒性机理、产毒基因以及利用产毒基因进行产毒蓝藻的分子生物学检测等,并对研究中存在的问题和未来的研究方向进行了展望,目的是为建立有毒水华蓝藻的预警机制、科学评价产毒蓝藻及其藻毒素的生态、环境危害及生态环境治理提供理论和技术支撑。     

微囊藻毒素去毒酶转基因模型的构建

根据已克隆的鲢鱼(Hypophthalmichthysmolitrix)微囊藻毒素去毒酶cDNA全序列设计特异引物,利用PCR方法获得鲢鱼微囊藻毒素去毒酶基因编码区,将该编码区与绿色荧光蛋白连接,分别构建融合表达载体pEGFP-N1-sGST和双顺反子表达载体pIRES2-EGFP-sGST。利用脂质体转染法将融合表达载体pEGFP-N1-sGST转染Hela细胞,60h后检测到绿色荧光基因表达;通过显微注射,将双顺反子表达载体pIRES2-EGFP-sGST注入斑马鱼(Daniorerio)受精卵,获得了转鲢鱼微囊藻毒素去毒酶基因斑马鱼,从而构建了微囊藻毒素去毒酶转基因模型。上述2种转基因模型的成功构建为进一步研究鲢鱼、鳙鱼(Aristichthysnobilis)、罗非鱼(Oreochromisnilotica)等淡水鱼类微囊藻毒素去毒酶基因调控元件、去毒分子机理及研发转基因鲢鱼、鳙鱼、罗非鱼等微囊藻毒素高效生物去毒器奠定了基础。     

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.     

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。     

运用分子生物学技术监测水环境中产毒蓝藻

有毒蓝藻在形成水华破坏水体环境的同时,也对人畜产生危害。运用分子生物学技术对水环境中产毒蓝藻进行监测,由于其简单、快速和经济等优点,逐渐成为各国学者关注的热点。本文从以下3个方面对其进行了综述:1)早期分子生物学技术在产毒蓝藻诊断中的运用;2)以产毒基因为靶标进行的产毒蓝藻诊断;3)运用基因芯片技术对产毒蓝藻进行检测。与传统的分子生物学技术相比,生物芯片技术具有体积小、重量轻、便于携带、分析自动快速、高通量等许多传统方法所不能比拟的优势。因此,该技术在蓝藻毒素检测中的运用必将给目前的产毒蓝藻的检测带来一场新的革命。     

一种快速检测微囊藻毒素mcyG基因的新技术——环介导恒温扩增

利用环介导恒温扩增(LAMP)技术,以微囊藻毒素(Microcystins,MCs)合成基因簇中的mcyG基因为靶序列,设计了1 套LAMP引物,建立了LAMP反应体系并进行灵敏度和特异性实验。结果表明mcyG基因的最低检测限为:24 cfu/mL,远低于常规PCR(Polymerase Chain Reaction)。整个检测过程仅需40 min,且可直接目测结果。特异性实验中, 13 株淡水常见水华蓝藻分属:色球藻属(Chroococcus)、念珠藻属(Nostoc)、鱼腥藻属(Anabaena)、束丝藻属(Aphanizomenon)、微囊藻属(Microcystis),其中10 株呈阳性反应, 3 株为阴性。在野外样品检测中,来自太湖与黄庆苗池塘的水样PCR检测显示阴性反应,而LAMP检测均呈阳性反应,提示此两处水样中可能含有产毒微囊藻,显示出了LAMP检测方法良好的野外检测和预警能力。综合上述,LAMP检测方法能够快速检测产微囊藻毒素的关键基因,且结果可视化。该方法简便、快捷、不依赖特殊检测设备,极具推广前景。     

利用PCR方法研究广州市景观湖产毒微囊藻的季节分布特点

为研究景观水体中产毒微囊藻的季节性分布特点,利用针对蓝藻和微囊藻的16srDNA、微囊藻毒素合成酶 mcyB 基因的部分核苷酸序列设计和筛选的特异性引物,对广州市内8个景观湖108份水样进行了冬季、夏季和秋季的二重及套式PCR的检测。结果显示,在冬季能被检测出的产毒微囊藻的阳性水样为42份,夏季为102份,而秋季为100份;阳性率分别为38.9%、94.4%、92.5%,产毒微囊藻在夏季和秋季阳性率高。结果表明在冬季、夏季和秋季均有产毒微囊藻分布;夏、秋季是广州市景观水体微囊藻污染的高峰季节,值得引起水文部门足够的重视。     

Competition for light between toxic and nontoxic strains of the harmful cyanobacterium Microcystis

The cyanobacterium Microcystis can produce microcystins, a family of toxins that are of major concern in water management. In several lakes, the average microcystin content per cell gradually declines from high levels at the onset of Microcystis blooms to low levels at the height of the bloom. Such seasonal dynamics might result from a succession of toxic to nontoxic strains. To investigate this hypothesis, we ran competition experiments with two toxic and two nontoxic Microcystis strains using light-limited chemostats. The population dynamics of these closely related strains were monitored by means of characteristic changes in light absorbance spectra and by PCR amplification of the rRNA internal transcribed spacer region in combination with denaturing gradient gel electrophoresis, which allowed identification and semiquantification of the competing strains. In all experiments, the toxic strains lost competition for light from nontoxic strains. As a consequence, the total microcystin concentrations in the competition experiments gradually declined. We did not find evidence for allelopathic interactions, as nontoxic strains became dominant even when toxic strains were given a major initial advantage. These findings show that, in our experiments, nontoxic strains of Microcystis were better competitors for light than toxic strains. The generality of this finding deserves further investigation with other Microcystis strains. The competitive replacement of toxic by nontoxic strains offers a plausible explanation for the gradual decrease in average toxicity per cell during the development of dense Microcystis blooms.     

Toxicity and toxins of natural blooms and isolated strains of Microcystis spp. (Cyanobacteria) and improved procedure for purification of cultures.

All samples of cyanobacterial blooms collected from 1986 to 1989 from Lake Kasumigaura, Ibaraki Prefecture, Japan, were hepatotoxic. The 50% lethal doses (LD50s) of the blooms to mice ranged from 76 to 556 mg/kg of body weight. Sixty-eight Microcystis cell clones (67 Microcystis aeruginosa and 1 M. viridis) were isolated from the blooms. Twenty-three strains (including the M. viridis strain) were toxic. However, the ratio of toxic to nontoxic strains among the blooms varied (6 to 86%). Microcystins were examined in six toxic strains. Five toxic strains produced microcystin-RR, -YR, and -LR, with RR being the dominant toxin in these strains. Another strain produced 7-desmethylmicrocystin-LR and an unknown microcystin. This strain showed the highest toxicity. Establishment of axenic strains from the Microcystis cells exhibiting extracellularly mucilaginous materials was successful by using a combination of the agar plate technique and two-step centrifugation.     

Toxicity and toxins of natural blooms and isolated strains of Microcystis spp. (Cyanobacteria) and improved procedure for purification of cultures

All samples of cyanobacterial blooms collected from 1986 to 1989 from Lake Kasumigaura, Ibaraki Prefecture, Japan, were hepatotoxic. The 50% lethal doses (LD50s) of the blooms to mice ranged from 76 to 556 mg/kg of body weight. Sixty-eight Microcystis cell clones (67 Microcystis aeruginosa and 1 M. viridis) were isolated from the blooms. Twenty-three strains (including the M. viridis strain) were toxic. However, the ratio of toxic to nontoxic strains among the blooms varied (6 to 86%). Microcystins were examined in six toxic strains. Five toxic strains produced microcystin-RR, -YR, and -LR, with RR being the dominant toxin in these strains. Another strain produced 7-desmethylmicrocystin-LR and an unknown microcystin. This strain showed the highest toxicity. Establishment of axenic strains from the Microcystis cells exhibiting extracellularly mucilaginous materials was successful by using a combination of the agar plate technique and two-step centrifugation.     

Variation between strains of the cyanobacterium Microcystis aeruginosa isolated from a Portuguese river

AIMS: The aim of this study was to investigate toxicological differences between strains of the cyanobacterium Microcystis aeruginosa isolated from a potable water supply in the north of Portugal over a 2-month period. METHODS AND RESULTS: Twenty-six strains of M. aeruginosa were isolated, grown in pure culture, and tested using a range of techniques including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), ELISA and a PCR procedure targeting the genes implicated in the production of toxic microcystins. There was considerable variation with respect to the amounts of microcystin produced by each of the strains as measured by ELISA, with values ranging from 0.02 to 0.53% dry weight. The results of the MALDI-TOF MS analysis demonstrated the presence of several chemically distinct forms of microcystin as well as aeruginosins, anabaenopeptins and several other unidentified peptide-like compounds. CONCLUSIONS: The growth of individual strains that comprise bloom populations, with unique 'chemotypes' can potentially be an important factor affecting the toxicity of bloom populations. Molecular probes, targeting the genes responsible for microcystin production were shown to be useful for distinguishing between toxic and nontoxic strains and showed good agreement with the results obtained from the other analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study show that the analysis of cyanobacterial bloom populations at the subspecies (strain) level can potentially provide important information regarding the toxin-producing potential of a cyanobacterial bloom and could be used as an 'early warning' for toxic bloom development.     

Quantitative real-time PCR for determination of microcystin synthetase e copy numbers for microcystis and anabaena in lakes

Cyanobacterial mass occurrences in freshwater lakes are generally formed by Anabaena, Microcystis, and Planktothrix, which may produce cyclic heptapeptide hepatotoxins, microcystins. Thus far, identification of the most potent microcystin producer in a lake has not been possible due to a lack of quantitative methods. The aim of this study was to identify the microcystin-producing genera and to determine the copy numbers of microcystin synthetase gene E (mcyE) in Lake Tuusulanj?rvi and Lake Hiidenvesi in Finland by quantitative real-time PCR. The microcystin concentrations and cyanobacterial cell densities of these lakes were also determined. The microcystin concentrations correlated positively with the sum of Microcystis and Anabaena mcyE copy numbers from both Lake Tuusulanj?rvi and Lake Hiidenvesi, indicating that mcyE gene copy numbers can be used as surrogates for hepatotoxic Microcystis and ANABAENA: The main microcystin producer in Lake Tuusulanj?rvi was Microcystis spp., since average Microcystis mcyE copy numbers were >30 times more abundant than those of ANABAENA: Lake Hiidenvesi seemed to contain both nontoxic and toxic Anabaena as well as toxic Microcystis strains. Identifying the most potent microcystin producer in a lake could be valuable for designing lake restoration strategies, among other uses.     

Detection of hepatotoxic Microcystis strains by PCR with intact cells from both culture and environmental samples

Microcystins are small hepatotoxic peptides produced by a number of cyanobacteria. They are synthesized non-ribosomally by multifunctional enzyme complex synthetases encoded by the mcy genes. Primers deduced from mcy genes were designed to discriminate between toxic microcystin-producing strains and non-toxic strains. Thus, PCR-mediated detection of mcy genes could be a simple and efficient means to identify potentially harmful genotypes among cyanobacterial populations in bodies of water. We surveyed the distribution of the mcyB gene in different Microcystis strains isolated from Chinese bodies of water and confirmed that PCR can be reliably used to identify toxic strains. By omitting any DNA purification steps, the modified PCR protocol can greatly simplify the process. Cyanobacterial cells enriched from cultures, field samples, or even sediment samples could be used in the PCR assay. This method proved sensitive enough to detect mcyB genes in samples with less than 2,000 Microcystis cells per ml. Its accuracy, specificity and applicability were confirmed by sequencing selected DNA amplicons, as well as by HPLC, ELISA and mouse bioassay as controls for toxin production of every strain used.     

Toxicity of Microcystis species isolated from natural blooms and purification of the toxin.

Microcystis strains (2 toxic and 18 nontoxic to mice) were isolated from toxic waterblooms that had been collected from Lake Kasumigaura, Ibaraki Prefecture, Japan, in August 1985. Thirteen of the strains (2 toxic and 11 nontoxic) were Microcystis aeruginosa, 2 (nontoxic) were Microcystis wesenbergii, and the other 5 were difficult to identify. Six (1 toxic and 4 nontoxic M. aeruginosa and 1 M. wesenbergii) of these 20 strains were established as axenic cultures. A toxic and axenic strain of M. aeruginosa, K-139, was used to study the relationship between growth conditions and toxicity. Cells in early-to-mid-log phase showed the highest toxicity (50% lethal dose, 7.5 mg of cells per kg of mouse), and maximum toxicity was not affected by growth temperatures between 22 and 30 degrees C. Purification and characterization of the toxins from K-139 cells were also conducted, and at least two toxins were detected. One of the toxins (molecular mass, 980 daltons) has not been reported previously. The main target of the toxin in mice was the liver. Marked congestion and necrosis in the parenchymal cells around the central veins of the liver were observed microscopically in specimens that had been prepared from the mice with acute toxicity after injection with the toxin.     

Effect of Nitrogen and Phosphorus on Growth of Toxic and Nontoxic Microcystis Strains and on Intracellular Microcystin Concentrations

The growth and intracellular microcystin concentration of two hepatotoxic and two nontoxic axenic Microcystis strains were measured in batch cultures with variable nitrogen (0.84-84 mg L(-1)) and phosphorus (0.05-5.5 mg L(-1)) concentrations. Growth was estimated by measuring dry weight, optical density, chlorophyll a, and cellular protein concentration. Microcystin concentrations in cells and in culture medium were measured by HPLC analysis. Both nontoxic strains needed less nutrients for their growth at low nutrient concentrations. With high nutrient concentrations the toxic strains grew better than the nontoxic strains. Growth and intracellular microcystin concentration did not correlate in the hepatotoxic strains. Multivariate regression analysis together with mathematical modeling revealed a significant interactive effect of nitrogen and phosphorus, which partly explains the controversial results obtained in previous studies. In this study we have shown that variation of nitrogen and phosphorus concentrations influence the growth and the microcystin production of Microcystis strains and that the strains differ in their response to nutrients. High levels of nitrogen and phosphorus in freshwaters may favor the growth of toxic Microcystis strains over nontoxic ones.     

Detection of toxigenicity by a probe for the microcystin synthetase A gene (mcyA) of the cyanobacterial genus Microcystis: comparison of toxicities with 16S rRNA and phycocyanin operon (Phycocyanin Intergenic Spacer) phylogenies

The relationship between toxigenicity and phylogeny within the cyanobacterial genus Microcystis is unclear. To investigate this issue, we have designed PCR primers for the N-methyltransferase (NMT) domain of the microcystin synthetase gene mcyA and have probed 37 Microcystis sp. cultures as well as several field samples. The NMT region was present in all 18 laboratory strains that gave positive reactions in the protein phosphatase inhibition assay for microcystin but was absent in 17 nontoxic strains. Two other nontoxic strains, one of which had previously been reported to produce microcystin, possessed the NMT region. Detection of NMT-specific DNA in field samples corresponded to periods of toxicity as assessed by protein phosphatase inhibition. The Microcystis strains formed a monophyletic cluster based on 16S rRNA gene sequences but comprised two groups with respect to phycocyanin intergenic spacer (PC-IGS) sequences. Toxic and nontoxic strains appeared to be erratically distributed within the PC-IGS and 16S rRNA trees. Sequence analysis of the NMT domain revealed two coherent groups. The genomic region immediately downstream of the mcyABC cluster in all 20 NMT-positive strains contained an open reading frame of unknown function (uma1) at a conserved distance from mcyC. All nontoxic strains also contained uma1, which is not cotranscribed with mcyABC. The consistent linkage of mcyC to uma1 suggests that mcyC has not been frequently transferred into nontoxic strains via any mechanism involving insertion at random chromosomal locations. These results are discussed with respect to various mechanisms that could explain the patchy distribution of toxigenicity among the various Microcystis clades.     

Comparative protein expression in different strains of the bloom-forming cyanobacterium Microcystis aeruginosa

Toxin production in algal blooms presents a significant problem for the water industry. Of particular concern is microcystin, a potent hepatotoxin produced by the unicellular freshwater species Microcystis aeruginosa. In this study, the proteomes of six toxic and nontoxic strains of M. aeruginosa were analyzed to gain further knowledge in elucidating the role of microcystin production in this microorganism. This represents the first comparative proteomic study in a cyanobacterial species. A large diversity in the protein expression profiles of each strain was observed, with a significant proportion of the identified proteins appearing to be strain-specific. In total, 475 proteins were identified reproducibly and of these, 82 comprised the core proteome of M. aeruginosa. The expression of several hypothetical and unknown proteins, including four possible operons was confirmed. Surprisingly, no proteins were found to be produced only by toxic or nontoxic strains. Quantitative proteome analysis using the label-free normalized spectrum abundance factor approach revealed nine proteins that were differentially expressed between toxic and nontoxic strains. These proteins participate in carbon-nitrogen metabolism and redox balance maintenance and point to an involvement of the global nitrogen regulator NtcA in toxicity. In addition, the switching of a previously inactive toxin-producing strain to microcystin synthesis is reported.