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Copy number of cyrJ and rpoC and extracellular CYN concentration in field samples collected during summer 2009 from Alange reservoir, Spain. Error bars are marked. n = 2.
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Cylindrospermopsin (CYN) is an alkaloid that causes hepatotoxicity, neurotoxicity and general cytotoxicity in vertebrates. It is currently gaining widespread attention after its reported appearance in water bodies around the world. A. ovalisporum is capable of CYN-production and can form toxic blooms when favorable environmental conditions are avai...
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... qPCR results were also compared with measurement of CYN concentrations in the water samples by LC-MS/ MS. As shown in Figure 2, the assay was able to detect and quantify the presence of A. ovalisporum in the sam- ples. cyrJ gene copy numbers increased from August to September and then decreased again to fall below the detection limit of our qPCR assay. ...
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... Campo et al. found that cyrJ is the gene suitable for designing primers and probes and established a Taqman qPCR assay for specific detection of toxigenic Aphanizomenon and Cylindrospermopsis. The presence of the cyrJ gene in cyanobacteria was in concordance with the toxin production, as revealed by testing 11 experimental strains [73]. Fergusson et al. designed primers out of cyrC regions encoding polyketide synthase and peptide synthetase and combined them into a multiplex PCR. ...
Although cyanobacteria are essential microorganisms on earth, some cyanobacteria produce toxins known as cyanotoxins, threatening humans and animals’ health. Hence, it is imperative to rapidly and accurately identify those toxic cyanobacteria. Unfortunately, traditional microscopic methods have limitations for accurate identification due to the lack of discernable morphological difference between toxic and non-toxic strains within the same cyanobacterial species or genus. In contrast, their genetic profiles are inherently conserved; therefore, nucleic acid-based assays can be more reliable for precise identification. Furthermore, molecular assays can provide high throughput and significantly reduce the turnaround time of test results. Such advantages make those assays a preferred method for rapid detection and early warning of potential toxicity. Toxigenic cyanobacterial species have synthetase genes (DNAs) for toxin production, which can be excellent marker genes. Numerous molecular assays targeting cyanotoxin synthetase genes have been developed for the identification of toxigenic cyanobacteria at various taxonomic levels. Polymerase chain reaction (PCR)-based assays are the most prevailing. Among different versions of PCR assays, the real-time quantitative PCR can be utilized to quantify the genes of interest in samples, fulfilling the purpose of both taxonomic recognition and biomass estimation. Reverse transcription (RT)-PCR assays can be used to detect transcripts (i.e., mRNAs) from toxin synthetase genes, probably enhancing the predictive value of PCR detection for toxin production from observed cyanobacterial species. Nevertheless, the utility of toxin synthetase gene- or its transcript-based PCR assays for routine cyanotoxin monitoring needs to be further evaluated on a large scale.
... We focused on water bodies in different regions of Slovenia (central Europe) and on three groups of cyanotoxins: microcystins, cylindrospermopsins and saxitoxins. We employed five previously published qPCR assays for detection of the microcystin- [15][16][17], cylindrospermopsin- [18] and saxitoxin-producing cyanobacteria [6]. Although the cyanotoxin potential is not neces-sarily linked to cyanotoxin concentration, we evaluated the correlation between the number of gene copies, microscopically determined cell number of potentially toxic species and cyanotoxin concentration. ...
... For our study, we have chosen previously published assays mcyE-Ana, mcyE-Mic, mcyE-Pla, cyrJ and sxtA, targeting microcystin-producers from genera Dolichospermum (ex Anabaena), Microcystis and Planktothrix, cylindrospermopsin-producers and saxitoxinproducers, respectively ( [6,[15][16][17][18]; Table 1). First, we evaluated the selected qPCR assays in terms of their specificity, sensitivity and robustness. ...
... In some of the assays, amplification efficiency was relatively low (Table 3). While in the original studies amplification efficiency exceeded 90% [6,16,18] for all evaluated assays, in our study that was the case only for assay sxtA. This difference might be caused by sequence variability of uncharacterised cyanobacterial cultures, which is even more significant between different geographical regions of sampling. ...
Due to increased frequency of cyanobacterial blooms and emerging evidence of cyanotoxicity in biofilm, reliable methods for early cyanotoxin threat detection are of major importance for protection of human, animal and environmental health. To complement the current methods of risk assessment, this study aimed to evaluate selected qPCR assays for detection of potentially toxic cyanobacteria in environmental samples. In the course of one year, 25 plankton and 23 biofilm samples were collected from 15 water bodies in Slovenia. Three different analyses were performed and compared to each other; qPCR targeting mcyE, cyrJ and sxtA genes involved in cyanotoxin production, LC-MS/MS quantifying microcystin, cylindrospermopsin and saxitoxin concentration, and microscopic analyses identifying potentially toxic cyanobacterial taxa. qPCR analyses detected potentially toxic Microcystis in 10 lake plankton samples, and potentially toxic Planktothrix cells in 12 lake plankton and one lake biofilm sample. A positive correlation was observed between numbers of mcyE gene copies and microcystin concentrations. Potential cylindrospermopsin- and saxitoxin-producers were detected in three and seven lake biofilm samples, respectively. The study demonstrated a potential for cyanotoxin production that was left undetected by traditional methods in both plankton and biofilm samples. Thus, the qPCR method could be useful in regular monitoring of water bodies to improve risk assessment and enable timely measures.
... In general, qPCR assays have been used either for 16S rDNA gene regions specific for cyanobacteria (Rinta-Kanto et al., 2005) or for gene loci specific for individual cyanobacterial genera known to produce cyanotoxins such as (i) the phycocyanin-intergenic spacer region (PC-IGS) for the genera Microcystis (Kurmayer & Kutzenberger, 2003) and Planktothrix (Ostermaier & Kurmayer, 2009;Kurmayer et al., 2011); (ii) the RNA polymerase gene loci rpoC for Raphidiopsis sp. (Fergusson & Saint, 2003;Rasmussen et al., 2008) and Chrysosporum (Aphanizomenon) ovalisporum (Campo et al., 2013); or (iii) the 16S rDNA for Microcystis sp. (Rinta-Kanto et al., 2005). ...
... A calibration curve based on defined cell concentrations or gene copy numbers is established by relating the known DNA concentrations to the threshold cycle of the diluted DNA extract. Similarly, qPCR assays have been developed for cylindrospermopsin synthesis genes (Rasmussen et al., 2008;Campo et al., 2013) and saxitoxin genes (Al-Tebrineh et al., 2010;Savela et al., 2015). For anatoxin synthesis genes, a qPCR approach was developed by Wang et al. (2015). ...
... bergii strains isolated from Lake Kinneret (Israel) contained aoaA/cyrA, aoaB/cyrB and aoaC/cyrC fragments but lacked the cyrJ gene, indicating that cyrJ is a suitable genetic marker for CYN producers (Alster et al., 2010;Ballot et al., 2011;Mihali et al., 2008). Accurate screening of CYN producers in cultured strains and environmental samples has been verified by PCR amplification of the cyrJ gene (Campo et al., 2013;Jiang et al., 2014;Mankiewicz-Boczek et al., 2012;Mazmouz et al., 2010). ...
... Quantitative real-time PCR (qPCR) can be used in this process, based on the assumption that the copy number of CYN biosynthesis genes reflects toxicity. Campo et al. (2013) first developed the TaqMan qPCR assay to detect CYN-producing Chr. ovalisporum in field samples. ...
The cyanotoxin cylindrospermopsin (CYN), a toxic metabolite from cyanobacteria, is of particular concern due to its cosmopolitan occurrence, aquatic bioaccumulation, and multi-organ toxicity. CYN is the second most often recorded cyanotoxin worldwide, and cases of human morbidity and animal mortality are associated with ingestion of CYN contaminated water. The toxin poses a great challenge for drinking water treatment plants and public health authorities. CYN, with the major toxicity manifested in the liver, is cytotoxic, genotoxic, immunotoxic, neurotoxic and may be carcinogenic. Adverse effects are also reported for endocrine and developmental processes. We present a comprehensive review of CYN over the past four decades since its first reported poisoning event, highlighting its global occurrence, biosynthesis, toxicology, removal, and monitoring. In addition, current data gaps are identified, and future directions for CYN research are outlined. This review is beneficial for understanding the ins and outs of this environmental pollutant, and for robustly assessing health hazards posed by CYN exposure to humans and other organisms.
... Previous studies have shown that the cyrJ gene encoding sulfotransferase is present only in CYN-producing strains and may be a good marker for CYN toxicity (Mihali et al. 2008;Mazmouz et al. 2010;Ballot et al. 2011). This gene has been employed to develop a two-step qPCR assay for detecting potential CYN-producing Aphanizomenon ovalisporum (Campo et al. 2013). A real-time PCR specifically targeting cyrJ gene may also be applicable to the monitoring and quantification of CYN-producing C. raciborskii in the future. ...
... In some studies, a real-time PCR was designed to detect both a cyr gene and a reference gene (rpoC1 or 16S rDNA) and thus CYN-producing cell numbers and the proportion of toxic and nontoxic strains of C. raciborskii were easily quantified. A positive correlation between cyr gene copy numbers and CYN concentrations was found in several field studies, indicating that the cyr gene copies in field samples are reliable at predicting the potential toxicity of C. raciborskii blooms (Orr et al. 2010;Al-Tebrineh et al. 2012;Campo et al. 2013;Zhang et al. 2014). ...
... In fact, the primers k18/m4 targeting cyrC, which have been originally applied to quantify toxic C. raciborskii in field samples, can also amplify CYN-producing Aphanizomenon and Anabaena (Fergusson and Saint 2003;Rasmussen et al. 2008;Orr et al. 2010;Moreira et al. 2011;Zhang et al. 2014). The cyrJ primers targeting Aphanizomenon also gave a positive amplification for CYN-producing C. raciborskii (Campo et al. 2013). In the present study, we have assumed that C. raciborskii was the only species that can produce CYN. ...
Cylindrospermopsin (CYN) is of great concern to human and animal health due to its potential toxicity. The cyanobacterium Cylindrospermopsis raciborskii is considered the most common cyanobacterial species that produces CYN. The discovery of the cyr gene cluster responsible for CYN biosynthesis allows us to develop molecular methods that detect and quantify potentially CYN-producing C. raciborskii. This paper describes the development of real-time PCR (qPCR) assays capable of quantifying the total and CYN-producing C. raciborskii in subtropical reservoirs of southern China. We designed primers and probes specifically targeting the rpoC1 and cyrJ genes of C. raciborskii, and enabling quantification of its total cell numbers and potentially toxic genotypes, respectively. The qPCR showed strong linearity between 10² and 10⁶ copies per reaction for both genes. This molecular method was validated against microscopic counting and a high correlation was found between them for quantifying C. raciborskii cell numbers in cyanobacterial cultures and water samples. Using qPCR, we detected potentially CYN-producing C. raciborskii in 34 of the 46 subtropical reservoirs of southern China, with the cyrJ/rpoC1 proportion ranging from 0.3 to 34.7%. CYN concentrations significantly correlated with both the copy numbers of cyrJ gene and the proportion of toxic C. raciborskii. Thus, the present real-time PCR method provides a reliable and faster method for estimating the potential toxicity of C. raciborskii blooms in this region. The wide distribution of potential CYN-producing C. raciborskii in the investigated subtropical reservoirs highlights the need for further monitoring.
... A TaqMan-based qPCR assay for quantifying A. ovalisporum CYN production was developed by Campo et al. [90]. This assay was able to discriminate CYN-producing A. ovalisporum strains from other Nostocales, such as C. raciborskii and A. bergii. ...
The wide distribution of cyanobacteria in aquatic environments leads to the risk of water contamination by cyanotoxins, which generate environmental and public health issues. Measurements of cell densities or pigment contents allow both the early detection of cellular growth and bloom monitoring, but these methods are not sufficiently accurate to predict actual cyanobacterial risk. To quantify cyanotoxins, analytical methods are considered the gold standards, but they are laborious, expensive, time-consuming and available in a limited number of laboratories. In cyanobacterial species with toxic potential, cyanotoxin production is restricted to some strains, and blooms can contain varying proportions of both toxic and non-toxic cells, which are morphologically indistinguishable. The sequencing of cyanobacterial genomes led to the description of gene clusters responsible for cyanotoxin production, which paved the way for the use of these genes as targets for PCR and then quantitative PCR (qPCR). Thus, the quantification of cyanotoxin genes appeared as a new method for estimating the potential toxicity of blooms. This raises a question concerning whether qPCR-based methods would be a reliable indicator of toxin concentration in the environment. Here, we review studies that report the parallel detection of microcystin genes and microcystin concentrations in natural populations and also a smaller number of studies dedicated to cylindrospermopsin and saxitoxin. We discuss the possible issues associated with the contradictory findings reported to date, present methodological limitations and consider the use of qPCR as an indicator of cyanotoxin risk.
... Interestingly, those non-producing Ch. ovalisporum strains contain other cyr genes (cyrA, cyrB, cyrC), a pattern that has also been observed in one non-producing C. issastchenkoi strain from New Zealand (Stü ken and Jakobsen, 2010). This has led to some authors to use cyrJ as the diagnostic marker in quantitative molecular probes (qPCR) aiming at detecting and quantifying toxic Ch. ovalisporum populations in the field (Campo et al., 2013). ...
... In these periphytic communities, there are important photosynthetic microorganisms, such as Cyanobacteria (blue algae), which are found in the community of periphytic algae or planktonic. These microorganisms become more populous because they are capable of forming massive growth of blooms with adverse effects on sanitation, which are aggravated when toxic cyanobacterial species are present [9]. However, the knowledge of their morphological variability (important for their identification) is still little known, which requires a thorough revision [10]. ...
This study aimed to perform the taxonomic survey of the species of periphytic Cyanobacteria in a lentic tropical environment, seeking to contribute to the knowledge on biodiversity and their dis-tribution. This study was conducted at the Samambaia Reservoir, which is located at Federal Uni-versity of Goiás, Goiânia, Central West region of Brazil. In general, the water of the Samambaia Reservoir is characterized as more turbid and the increased biochemical oxygen demand as well as a higher concentration of coliforms in the rainy season. Twenty six samples were collected, ten in the dry season (09/2010 and 11/2010; 07/2012) and ten in the rainy season (01/2011 and 03/2011; 01/2013). Periphyton was collected from stems of aquatic plants (Cyperaceae), which were in the marginal area of the reservoir. With the floristic survey, 38 taxa of Cyanobacteria were identified in periphyton of the Samambaia Reservoir. Phormidium (family Phormidiaceae) and Aphanocapsa (Merismopediaceae) are among genera with the highest species richness. Regarding morphological types, many species of filamentous cyanobacteria were identified, followed by colonial taxa. In relation to the frequency of occurrence and the seasonal period, 14 taxa were classified in the rare category, 12 in common category and 12 in constant category. Regarding the seasonal period, Cyanophyceae were the greatest wealth in the rainy season (97.4%).
... CYN-producing strains of Aph. ovalisporum have so far been identified only in limited locations including Spain, Italy, Israel (Lake Kinneret), Florida, United States and Queensland, Australia (Shaw et al., 1999; Quesada et al., 2006; Yilmaz et al., 2008; Messineo et al., 2010; Campo et al., 2013). It has been suggested that in Australia, this species may be a potentially more important producer of CYN than C. raciborskii (Shaw et al., 1999 ). ...
Despite a significant interest in cyanotoxins over recent decades, their biological role is still poorly elucidated. Cylindrospermopsin (CYN) is a cyanobacterial metabolite that is globally identified in surface fresh- and brackish waters and whose producers are observed to spread throughout different climate zones. This paper provides a comprehensive review of the characteristics and global distribution of CYN-producing species, the variety of their chemotypes and the occurrence of strains which, while incapable of toxin synthesis, are able to produce other bioactive compounds including those that are hitherto unknown and yet to be identified. Environmental conditions that can trigger CYN production and promote growth of CYN-producers in aquatic ecosystems are also discussed. Finally, on the basis of existing experimental evidence, potential ecological role(s) of CYN are indicated. It is eventually concluded that CYN can be at least partially responsible for the ecological success of certain cyanobacteria species.
... Future works should ultimately focus on seeking diagnostic genes present only in PSP producers, similar to the case of the gene cyrJ found only in CYN-producing strains but not in non-producers containing other cyr genes (Mihali et al., 2008;Ballot et al., 2011). In this sense, molecular tools combining fast evolving genes (e.g., cpcBA-IGS, rpoC1) with one or several toxin-biosynthesis genes, as already proposed for CYN producers (Campo et al., 2013), might be a feasible option to monitor STX-producing freshwater cyanobacteria at the species level. ...