Multiplex PCR for detection of microcystins-producing cyanobacteria from freshwater samples.
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 mcyA-cd, mcyAB, and mcyB 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.
Valério
Elisabete
E
Faculdade de Ciências, Universidade de Lisboa, Centro de Biodiversidade, Genómica Integrativa e Funcional (BioFIG), Edifício ICAT, Campus da FCUL, Campo Grande, Lisbon, Portugal.
Chambel
Lélia
L
Paulino
Sérgio
S
Faria
Natália
N
Pereira
Paulo
P
Tenreiro
Rogério
R
D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D000458:Cyanobacteria / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004784:Environmental Monitoring / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D005798:Genes, Bacterial; D052998:Microcystins / Q000096:biosynthesis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014871:Water Microbiology / Q000592:standards; D014881:Water Supply / Q000592:standards
D001427:Bacterial Toxins; D004269:DNA, Bacterial; D052998:Microcystins