Polymerase chain reaction (PCR) detection of the predominant microcystin-producing genotype of cyanobacteria in Mozambican lakes (original) (raw)
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DETECTION OF POTENTIALLY TOXIC MICROCYSTISAND CYANOBACTERIA BY MOLECULAR METHOD IN CÔTE d'IVOIRE
2015
The cyanobacteria or blue-green algae are natural inhabitants of water bodies. The eutrophication due to anthropogenic pressure favors the massive development of strains of cyanobacteria, some of which are potentially toxic. The main goal of this work was to demonstrate for the first time in Ivory Coast the presence of toxicity gene of cyanobacteria from cyanobacteria culture. Water used by the population for their different needed were sampled in different cities of the country. Several genera known for their ability to produce toxic were highlighted including Microcystis, anabeana, oscillatoria, plancktothrix. Oscillatoria was the most isolated genus with 93% of presence in the samples. The pcr has allowed us to confirm the existence of the gene mcy encoding microcystin in Microcystis. PCR identified the mcygène of microcystis in all the sample (100%) while microcystis was found only 86% by microscopy. The contribution of chromatographic and ELISA techniques will ultimately confirm their toxin production or not.
Occurrence of microcystin-producing cyanobacteria in Ugandan freshwater habitats
Environmental Toxicology, 2009
Microcystins (MCs) are cyclic heptapeptides that are the most abundant toxins produced by cyanobacteria in freshwater. The phytoplankton of many freshwater lakes in Eastern Africa is dominated by cyanobacteria. Less is known, however, on the occurrence of MC producers and the production of MCs. Twelve Ugandan freshwater habitats ranging from mesotrophic to hypertrophic conditions were sampled in May and June of 2004 and April of 2008 and were analyzed for their physico-chemical parameters, phytoplankton composition, and MC concentrations. Among the group of the potential MC-producing cyanobacteria, Anabaena (0 -10 7 cells ml −1 ) and Microcystis (10 3 -10 7 cells ml −1 ) occurred most frequently and dominated in eutrophic systems. A significant linear relationship (n = 31, r 2 = 0.38, p < 0.001) between the Microcystis cell numbers and MC concentration (1.3-93 fg of MC cell −1 ) was observed. Beside [MeAsp 3 , Mdha 7 ]-MC-RR two new microcystins, [Asp 3 ]-MC-RY and [MeAsp 3 ]-MC-RY were isolated and their constitution assigned by LC-MS 2 .
Harmful Algae, 2003
One of the most serious problems related to water eutrophication is the occurrence of increasingly frequent blooms of toxic cyanobacteria in freshwater ecosystems. Microcystin (MCYST) molecular markers may be used for the detection of toxic cyanobacteria, both cultivated strains and environmental samples, independently of their taxonomic category and production of the toxin at the moment of analysis. Sixty Microcystis spp. strains from 15 water reservoirs of south, southeastern and northeastern Brazil were analyzed by polymerase chain reaction (PCR) with oligonucleotide primers for mcyB gene of the operon that encodes a microcystin synthetase. It was found out that the presence of a unique amplified product of approximately 780 bp in 18 strains, indicated the presence of the microcystin-producing genotype. There was correspondence between the presence of the mcyB gene and microcystin determined by ELISA. Eight reservoirs contained toxic strains, two of these reservoirs being used mainly for public water supply. The coexistence of a mixture of toxic and non-toxic genotypes in populations of several reservoirs was found. Thus, it is evident that Microcystis populations present in blooms compose a mosaic, with genetically different individuals within the same population, each one, possibly, with its own tolerance to environmental factors and with distinct toxicity potential.
Microcystin-producing genotypes from cyanobacteria in Brazilian reservoirs
Environmental Toxicology, 2012
designed from Brazilian cyanobacteria for the detection of microcystinproducing genotypes in 27 environmental samples from water reservoirs and 11 strains of Microcystis. Microcystins were found using HPLC in all 11 strains and 19 of the environmental samples. The new oligonucleotide primers amplified fragments of microcystin-producing genes, including the eight environmental samples in which no microcystins were detected by HPLC, but which presented amplified fragments, thereby demonstrating the existence of microcystin-producing genes. The new oligonucleotide primers exhibited better specificity when used with environmental samples and were more reliable in comparison with those described in the literature (mcyB-FAA/RAA and mcyA-Cd/FR), which generate false-negative results. The better performance of these new oligonucleotide primers underline the need for designing molecular markers that are well fitted to the regional biological diversity. As this is a fast predictive technique for determining the presence or absence of microcystins, it could be used either alone or in conjunction with other techniques, such as the screening of samples to be sent for quantitative toxicological analysis using HPLC, thereby reducing monitoring cost and time.
Biokemistri, 2006
Toxic cyanobacteria found in eutrophic, municipal and residential water supplies are an increasing environmental hazard in South Africa. Cyanobacteria produce lethal toxins, and domestic and wild animal deaths are caused by drinking water contaminated by these toxins. Among the species causing death of livestock, blooms of Microcystis aeruginosa are the most common in South Africa. More than 65 microcystins have been isolated to date and they are the most abundant cyanobacterial toxins. Hazards to human health may result from chronic exposure via contaminated water supplies. Microcystins are powerful tumour promoters and inhibitors of protein phosphatase 1 and 2A and they are suspected to be involved in the promotion of primary liver cancer in humans. In this minireview, we discuss the significance of toxic cyanobacteria in South Africa as well as the detection of potential microcystin-producing cyanobacteria strains in South African reservoirs with a mcyB molecular marker. It would be of economic and public health value to be able to detect early stage blooms of cyanobacteria, especially if it is on a sufficiently timely basis for municipalities and recreation facilities to implement a response plan.
2021
Water quality assessment relies mostly on physico-chemical based characterization however, eutrophication and climate change advocate the abundance of toxic microcystins (MCs) producing cyanobacteria as emerging bio-indicator. In the present study, a spatial-temporal analysis was carried out at ten sampling sites of Prayagraj and Varanasi during June 2017 and March 2018 to determine the Ganga River water quality using physico-chemical parameters, cyanobacteria diversity, detection of MCs producing strains and MC-LR equivalence. Coliform bacteria, COD, NO3-N and phosphate are the significant contaminated parameters favoring the growth of putative MCs producing cyanobacteria. National Sanitation Foundation WQI (NSFWQI) indicates water quality, either bad or medium category at sampling points. The morphological analysis confirms the occurrence of diverse cyanobacterial genera such as Microcystis, Anabaena, Oscillatoria, Phormidium etc. PCR amplification affirmed the presence of toxic m...
Journal of Microbiological Methods, 2007
The application of quantitative real time PCR has been proposed for the quantification of toxic genotypes of cyanobacteria. We have compared the Taq Nuclease Assay (TNA) in quantifying the toxic cyanobacteria Microcystis sp. via the intergenic spacer region of the phycocyanin operon (PC) and mcyB indicative of the production of the toxic heptapeptide microcystin between three research groups employing three instruments (ABI7300, GeneAmp5700, ABI7500). The estimates of mcyB genotypes were compared using (i) DNA of a mcyB containing strain and a non-mcyB containing strain supplied in different mixtures across a low range of variation (0-10% of mcyB) and across a high range of variation (20-100%), and (ii) DNA from field samples containing Microcystis sp. For all three instruments highly significant linear regression curves between the proportion of the mcyB containing strain and the percentage of mcyB genotypes both within the low range and within the high range of mcyB variation were obtained. The regression curves derived from the three instruments differed in slope and within the high range of mcyB variation mcyB proportions were either underestimated (0-50%) or overestimated (0-72%). For field samples cell numbers estimated via both TNAs as well as mcyB proportions showed significant linear relationships between the instruments. For all instruments a linear relationship between the cell numbers estimated as PC genotypes and the cell numbers estimated as mcyB genotypes was observed. The proportions of mcyB varied from 2-28% and did not differ between the instruments. It is concluded that the TNA is able to provide quantitative estimates on mcyB genotype numbers that are reproducible between research groups and is useful to follow variation in mcyB genotype proportion occurring within weeks to months.
Environmental Toxicology, 2002
In Morocco, the occurrence of toxic cyanobacteria blooms is confirmed in some water bodies used for recreational and/or as drinking water reservoirs. According to WHO recommendations, the establishment of a monitoring program for microcystins is a necessity. This paper presents toxicological studies of 19 toxic cyanobacteria strains of Microcystis, Synechocystis, Pseudanabaena, and Oscillatoria. These strains were isolated from various water bodies including natural lakes, reservoirs, and ponds located in central regions of Morocco. The isolation, culture, and biomass production of these strains was made on Z8 or BG13 media under laboratory controlled conditions. The hepatotoxicity of cyanobacterial lyophilized material was confirmed by mouse bioassays. The amount of microcystins produced by each strain was determined by the enzyme-linked immunosorbent assay (ELISA). The detection and identification of microcystin variants was performed by high performance liquid chromatography (HPLC) with photodiode array detection. Almost all strains showed medium to high toxicity, the estimated LD50 i.p mice bioassay ranged between 28 to 350 mg/kg body weight. The concentrations of microcystins varied between 2.16 to 944 μg/g and 26.8 to 1884 μg/g dry weight determined by ELISA and HPLC, respectively. The screening of bloom-forming and microcystin producer cyanobacteria strains in these fresh water bodies leads us to propose the need for the establishment of a survey of cyanobacteria and a cyanotoxin-monitoring program. © 2002 by Wiley Periodicals, Inc. Environ Toxicol 17: 32–39, 2002
Multiplex PCR for detection of microcystins-producing cyanobacteria from freshwater samples
Environmental Toxicology, 2009
The aim of this study was to develop a PCR-based method of gene-directed multiplex PCR to rapidly identify microcystins producing cyanobacteria, regardless of their taxa, that could be applied in routine freshwater monitoring. Instead of using the amplification of only one or two mcy gene fragments, a multiplex PCR that simultaneously amplifies mcyA-cd, mcyAB, and mcyB fragments of the microcystin gene cluster was validated with DNA from 124 cyanobacterial isolates and applied in 37 environmental samples. The toxicological status of the isolates was assessed by high-performance liquid chromatography also used as the “gold standard” for the evaluation of multiplex mcy genes-based PCR, where a sensitivity of 92.3% and a specificity of 100% have been obtained. For the environmental samples, a rapid protocol for their direct use in the PCR reaction has been developed and, by using ELISA results as “gold standard” for the presence of microcystins in these samples, a sensitivity of 80% and a specificity of 100% were achieved, showing that this multiplex PCR test is a rapid, reliable, and economical way of assessing the microcystin-producing potential of cyanobacteria in freshwaters, regardless of their taxa or microcystins variant produced. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.
Detection and monitoring toxigenicity of cyanobacteria by application of molecular methods
Environmental Toxicology, 2006
The aim of this study was early genetic identification of microcystin-producing cyanobacteria and monitoring their toxigenicity by determining toxin concentrations in three Polish lakes throughout the summer of 2004. The assessment of cyanobacterial blooms was carried out in shallow, eutrophic water bodies: Lake Jeziorak, Lake Bninskie, and Sulejow Reservoir. Samples for DNA, phycological, and toxin analyses were collected from July till October. Molecular analysis of the 16S rRNA region was used to detect cyanobacteria in water samples. The microscopic analysis was performed to investigate seasonal variation of phytoplankton. Cyanobacteria, with domination by Microcystis, Planktothrix, and Planktolyngbya were detected during the whole monitoring period in Sulejow Reservoir, Lake Bninskie, and Lake Jeziorak, respectively. The presence and identification of toxic strains in water bodies was studied by PCR amplification of mcy genes in the microcystis synthesis pathway. The presence of the mcyA, mcyB, mcyD, and mcyE genes in water samples indicated the genetic potential to produce microcystins. Toxicity of water samples and microcystin concentrations were established by PPIA and HPLC, respectively. The maximum concentration of microcystins was 11.13 g/L and 4.67 g/L in samples dominated by P. agardhii and M. aeruginosa, respectively. Molecular analysis showed that toxigenic strains of cyanobacteria occurred in the three lakes throughout the summer season.