Abstract
Blooms of microcystin-producing cyanobacteria are a problem worldwide. Microcystin is a liver hepatotoxin commonly found in bodies of water and is produced mainly by the genus Microcystis. The aim of the present study was to develop and assess a competitive PCR method for the quantification of toxic and non-toxic Microcystis cells using the cpcBA and mcyB genes, which are respectively involved in the formation of phycocyanin and biosynthesis of microcystin. For the acquisition of competitor DNA, amplification sequences were carried out of the “cell DNA equivalent” of microcystin-producing (BCCUSP18) and non-microcystin-producing (BCCUSP03) strains of Microcystis spp. using primers described in the literature as well as others designed for the present study. The method was successfully developed, as competitor DNA was constructed and co-amplified with the target DNA. Competitive PCR proved to be useful in quantifying toxic and non-toxic cells of Microcystis spp. strains, representing a helpful methodology tool to study isolated toxin-producing cyanobacteria.
Highlights
Cyanobacterial blooms have become increasingly frequent in freshwater systems worldwide, causing countless adverse effects on public health and the environment
The aim of the present study was to develop and assess a competitive PCR method for the quantification of toxic and non-toxic Microcystis cells using the cpcBA and mcyB genes, which are respectively involved in the formation of phycocyanin and biosynthesis of microcystin
For the acquisition of competitor DNA, amplification sequences were carried out of the “cell DNA equivalent” of microcystin-producing (BCCUSP18) and non-microcystin-producing (BCCUSP03) strains of Microcystis spp. using primers described in the literature as well as others designed for the present study
Summary
Cyanobacterial blooms have become increasingly frequent in freshwater systems worldwide, causing countless adverse effects on public health and the environment. One of the greatest problems is that these organisms produce toxins with hepatotoxic and neurotoxic effects [1,2,3] These toxins are released in water through cell lysis and remain dissolved for varying periods of time. The monitoring of cyanobacteria in public supply reservoirs could be made easier by applying quicker methods able to overcome the difficulty in cell visualization in colonial individuals. This requires the development and employment of sensitive methods that can guarantee the detection and quantification of these cyanobacteria at low cost. The objective of this study was to develop and investigate the applicability and efficiency of the competitive PCR method to quantify toxic and non-toxic Microcystis cells. The accuracy of the competitive PCR technique was later assessed against a cell count by optical microscope
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