Detecção molecular quantitativa de cianobactérias hepatotóxicas através de PCR competitiva
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Data
2010-03-26
Autores
Barros, Selma Gouvêa de [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Florações de cianobactérias tóxicas são atualmente um problema mundial devido a produção de toxinas. A microcistina é uma hepatotoxina comumente encontrada em corpos d´água e é produzida principalmente pelo gênero Microcystis. Recentemente a identificação, clonagem e seqüenciamento do agrupamento de genes responsáveis pela codificação da sintetase de microcistina (MS) tornaram possível a abordagem molecular na detecção de populações tóxicas baseada na técnica de PCR (Polymerase Chain Reaction). A técnica de PCR tornou-se viável e útil pelo fato do agrupamento de genes da sintetase de microcistina estar presente apenas em organismos tóxicos e pela ausência de diferenças morfológicas entre aqueles tóxicos e não tóxicos. Este estudo objetivou desenvolver e avaliar a técnica de PCR competitiva para quantificação de células de Microcystis tóxicas e não tóxicas utilizando os genes cpcBA e mcyB envolvidos respectivamente, na formação da ficocianina e biossíntese da MS. Testou-se a hipótese de que a PCR competitiva pode ser utilizada como metodologia de quantificação de células tóxicas e não tóxicas de Microcystis em substituição a contagem direta de células por microscopia óptica para atender a Portaria do Ministério da Saúde 518/2004. Para obtenção de DNA competidores foram realizadas amplificações seqüenciais de “células DNA equivalente” das linhagens tóxica (BCCUSP18) e não tóxica (BCCUSP03) de Microcystis spp. utilizando primers descritos na literatura, bem como aqueles desenhados para este estudo. Avaliaram-se os DNA competidores amplificando-os com células DNA equivalente das linhagens. Após determinada a quantidade mais próxima de DNA competidores para quantificar 2,0 x 104 células, realizou-se a PCR competitiva com células DNA equivalente de uma amostra ambiental (reservatório de Duas Unas, PE)...
Blooms of microcystin-producing cyanobacteria are a problem worldwide. Mycrocystin is a liver hepatotoxin commonly found in bodies of water and is produced mainly by the genus Microcystis. The recent identification, cloning and sequencing of the genes responsible for coding microcystin synthetase (MS) has allowed a molecular approach to the detection of micocystin-producing populations based on the Polymerase Chain Reaction (PCR) method. Considering the lack of morphological differences between microcystin-producing and non-microcystin-producing organisms, PCR is viable and useful, as the cluster of microcystin synthetase genes is found in only microcystinproducing organisms. The aim of the present study was to develop and assess an 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 MS. The hypothesis was that competitive PCR could be used as a quantification method for toxic and non-toxic Microcystis cells, replacing the direct cell count under an optical microscope, while fulfilling Brazilian Ministry of Health Ordinance 518/2004. For the acquisition of competitor DNA, sequences amplifications 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. Competitor DNA was assessed by amplifying “cells DNA equivalent” of the strains. After determining the quantity closest to the competitor DNA for quantifying 2.0 x 104 cells, competitive PCR was carried out with “cells DNA equivalent” from an environmental sample (Duas Unas Reservoir, PE, Brazil). The constructed competitor cpcBA quantified 1.45 x 104 cells (R2= 0.989)... (Complete abstract click electronic access below)
Blooms of microcystin-producing cyanobacteria are a problem worldwide. Mycrocystin is a liver hepatotoxin commonly found in bodies of water and is produced mainly by the genus Microcystis. The recent identification, cloning and sequencing of the genes responsible for coding microcystin synthetase (MS) has allowed a molecular approach to the detection of micocystin-producing populations based on the Polymerase Chain Reaction (PCR) method. Considering the lack of morphological differences between microcystin-producing and non-microcystin-producing organisms, PCR is viable and useful, as the cluster of microcystin synthetase genes is found in only microcystinproducing organisms. The aim of the present study was to develop and assess an 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 MS. The hypothesis was that competitive PCR could be used as a quantification method for toxic and non-toxic Microcystis cells, replacing the direct cell count under an optical microscope, while fulfilling Brazilian Ministry of Health Ordinance 518/2004. For the acquisition of competitor DNA, sequences amplifications 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. Competitor DNA was assessed by amplifying “cells DNA equivalent” of the strains. After determining the quantity closest to the competitor DNA for quantifying 2.0 x 104 cells, competitive PCR was carried out with “cells DNA equivalent” from an environmental sample (Duas Unas Reservoir, PE, Brazil). The constructed competitor cpcBA quantified 1.45 x 104 cells (R2= 0.989)... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Alga, Cianobacteria, Microcystis, PCR competitiva, Genes mcyB, Genes cpcBA, Cyanobacteria, Microcystis, Competitive PCR
Como citar
BARROS, Selma Gouvêa de. Detecção molecular quantitativa de cianobactérias hepatotóxicas através de PCR competitiva. 2010. 53 f. Dissertação (mestrado) - Universidade Estadual Paulista, Instituto de Biociências de Rio Claro, 2010.