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Multiplex PCR for detection of microcystins‐producing cyanobacteria from freshwater samples
Author(s) -
Valério Elisabete,
Chambel Lélia,
Paulino Sérgio,
Faria Natália,
Pereira Paulo,
Tenreiro Rogério
Publication year - 2010
Publication title -
environmental toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.813
H-Index - 77
eISSN - 1522-7278
pISSN - 1520-4081
DOI - 10.1002/tox.20502
Subject(s) - microcystin , cyanobacteria , multiplex , multiplex polymerase chain reaction , biology , polymerase chain reaction , gene , microbiology and biotechnology , genetics , bacteria
Abstract 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 mcy A‐cd, mcy AB, and mcy B 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.