Development and field testing of a real-time PCR assay for cylindrospermopsin-producing cyanobacteria

Rasmussen, J. Paul; Giglio, Steven; Monis, Paul; Campbell, Rebecca; Saint, Christopher P.

doi:10.1111/j.1365-2672.2007.03676.x

Cited by 94 publications

(62 citation statements)

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“…Since portable real-time PCR instruments have been shown to be field applicable (35), it is conceivable that during future monitoring, those measurements will be confirmed directly on site subsequent to the filtration of water samples and the rapid extraction of the DNA from filters according to previously established protocols (41). The results would then be directly forwarded to the respective authorities and distributed to the public via websites, newspapers, and radio (9).…”

Section: Discussionmentioning

confidence: 99%

“…Nevertheless, the real-time PCR technique is the only quantitative technique available for estimating the proportion of potential toxin-producing genotypes in water. The development of automated and field-applicable real-time PCR methods (e.g., see reference 35), in particular, may contribute to a more widespread integration of real-time PCR into routine monitoring programs in the future.…”

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confidence: 99%

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Application of Real-Time PCR To Estimate Toxin Production by the Cyanobacterium Planktothrix sp

Ostermaier

Kurmayer

2010

Appl Environ Microbiol

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Quantitative real-time PCR methods are increasingly being applied for the enumeration of toxic cyanobacteria in the environment. However, to justify the use of real-time PCR quantification as a monitoring tool, significant correlations between genotype abundance and actual toxin concentrations are required. In the present study, we aimed to explain the concentrations of three structural variants of the hepatotoxin microcystin ( During the last decade, genetic methods have significantly increased our understanding of the distribution of genes that are involved in the production of toxins within cyanobacteria that occur in fresh and brackish water (45). Although genetic methods can indicate only the potential risk of toxin synthesis and do not provide information about the actual toxin concentrations, quantitative real-time PCR has been increasingly applied for monitoring the toxin-producing genotypes of cyanobacteria in water (26,33,44). The development of real-time PCR methods was driven primarily by its potential (i) as an early-warning tool as well as to monitor toxin-producing cyanobacteria and (ii) to identify those factors that lead to a dominance/repression of toxin-producing genotypes versus nontoxic genotypes. For the first aim, it is essential that the abundance of toxin-producing cyanobacteria can be related to the concentration of the respective toxic substance in water. A few studies showed that the concentration of certain toxic genotypes was linearly related to the respective toxin concentrations, e.g., for the most common group of hepatotoxins, the microcystins (MCs) (7,12,14), and for the related nodularin (19). Both microcystins and nodularins are known to be potent inhibitors of eukaryotic protein phosphatases 1 and 2A, resulting in a health hazard to humans and the environment (9). In contrast, no correlation was found (37, 50), or even the opposite was reported, by other studies, i.e., that the measurement of microcystin-producing genotypes is not a satisfactory method for use in monitoring programs in order to predict the toxic risk associated with cyanobacterial proliferation (3). For microcystins, these contrasting results may be due to several reasons: (i) several genera producing microcystins frequently coexist in water bodies, and therefore, not all microcystin producers may have been identified; (ii) the semilogarithmic calibration curves limit the accuracy in estimations of genotype numbers and proportions (for example, the only laboratory comparison carried out so far revealed that among the three laboratories tested, the proportions of toxic genotypes were overestimated or underestimated by 0 to 72% and 0 to 50%, respectively [42]); and (iii) inactive mutants that contain the respective genes, however, which have been inactivated in toxin production through the insertion of transposable elements, may co-occur and decrease toxin production in a given population (6). Nevertheless, the real-time PCR technique is the only quantitative technique available for estimating the proportion of...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Application of Real-Time PCR To Estimate Toxin Production by the Cyanobacterium Planktothrix sp

Ostermaier

Kurmayer

2010

Appl Environ Microbiol

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“…Since the entire gene sequences and flanking regions of these fragments are not available, it can only be speculated that they represent signs of HGT events. In this light, it would also be valuable to sequence the aoa gene fragments and flanking regions found in three non-CYN -producing strains, which are thought to represent natural deletion mutants (Rasmussen et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

The cylindrospermopsin gene cluster of Aphanizomenon sp. strain 10E6: organization and recombination

Stüken

Jakobsen

2010

Microbiology

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Cylindrospermopsin (CYN), a potent hepatoxin, occurs in freshwaters worldwide. Several cyanobacterial species produce the toxin, but the producing species vary between geographical regions. Aphanizomenon flos-aquae, a common algae species in temperate fresh and brackish waters, is one of the three well-documented CYN producers in European waters. So far, no genetic information on the CYN genes of this species has been available. Here, we describe the complete CYN gene cluster, including flanking regions from the German Aphanizomenon sp. strain 10E6 using a full genome sequencing approach by 454 pyrosequencing and bioinformatic identification of the gene cluster. In addition, we have sequenced a~7 kb fragment covering the genes cyrC (partially), cyrA and cyrB (partially) of the same gene cluster in the CYN-producing Aphanizomenon sp. strains 10E9 and 22D11. Comparisons with the orthologous gene clusters of the Australian Cylindrospermopsis raciborskii strains AWT205 and CS505 and the partial gene cluster of the Israeli Aphanizomenon ovalisporum strain ILC-146 revealed a high gene sequence similarity, but also extensive rearrangements of gene order. The high sequence similarity (generally higher than that of 16S rRNA gene fragments from the same strains), atypical GCcontent and signs of transposase activities support the suggestion that the CYN genes have been horizontally transferred.

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“…Genes encoding the biosynthesis of other cyanobacterial toxins, e.g. CYN and STXs, have been detected in several non-toxin-producing cyanobacteria (Wood et al 2007;Rasmussen et al 2008;Ballot et al 2010b). Various mechanisms, such as horizontal gene transfer, mutations, insertions and deletions, have been proposed as explanations for non-toxin-producing cyanobacteria possessing parts of toxinencoding gene clusters (Christiansen et al 2008;ToomingKlunderud et al 2008;Moustafa et al 2009).…”

Section: R Curvata From Hartbeespoort Dam Than To Cylindrospermopsismentioning

confidence: 99%

Diversity of cyanobacteria and cyanotoxins in Hartbeespoort Dam, South Africa

Ballot

Sandvik

Rundberget

et al. 2014

Mar. Freshwater Res.

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Abstract. The South African Hartbeespoort Dam is known for the occurrence of heavy Microcystis blooms. Although a few other cyanobacterial genera have been described, no detailed study on those cyanobacteria and their potential toxin production has been conducted. The diversity of cyanobacterial species and toxins is most probably underestimated. To ascertain the cyanobacterial composition and presence of cyanobacterial toxins in Hartbeespoort Dam, water samples were collected in April 2011. In a polyphasic approach, 27 isolated cyanobacterial strains were classified morphologically and phylogenetically and tested for microcystins (MCs), cylindrospermopsin (CYN), saxitoxins (STXs) and anatoxin-a (ATX) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and screened for toxin-encoding gene fragments. The isolated strains were identified as Sphaerospermopsis reniformis, Sphaerospermopsis aphanizomenoides, Cylindrospermopsis curvispora, Raphidiopsis curvata, Raphidiopsis mediterrranea and Microcystis aeruginosa. Only one of the Microcystis strains (AB2011/53) produced microcystins (35 variants). Forty-one microcystin variants were detected in the environmental sample from Hartbeespoort Dam, suggesting the existence of other microcystin producing strains in Hartbeespoort Dam. All investigated strains tested negative for CYN, STXs and ATX and their encoding genes. The mcyE gene of the microcystin gene cluster was found in the microcystin-producing Microcystis strain AB2011/53 and in eight non-microcystin-producing Microcystis strains, indicating that mcyE is not a good surrogate for microcystin production in environmental samples.

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Development and field testing of a real-time PCR assay for cylindrospermopsin-producing cyanobacteria

Cited by 94 publications

References 50 publications

Application of Real-Time PCR To Estimate Toxin Production by the Cyanobacterium Planktothrix sp

Application of Real-Time PCR To Estimate Toxin Production by the Cyanobacterium Planktothrix sp

The cylindrospermopsin gene cluster of Aphanizomenon sp. strain 10E6: organization and recombination

Diversity of cyanobacteria and cyanotoxins in Hartbeespoort Dam, South Africa

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