Dual quantitative PCR assay for identification and enumeration of Karlodinium veneficum and Karlodinium armiger combined with a simple and rapid DNA extraction method
Karlodinium is a dinoflagellate genus responsible for massive fish mortality events worldwide. It is commonly found in Alfacs Bay (NW Mediterranean Sea), where the presence of two Karlodinium species (K. veneficum and K. armiger) with different toxicities has been reported. Microscopy analysis is not able to differentiate between these two species. Therefore, new and rapid methods that accurately and specifically detect and differentiate these two species are crucial to facilitate routine monitoring, to provide early warnings and to study population dynamics. In this work, a quantitative real-time PCR (qPCR) method to detect and enumerate K. veneficum and K. armiger is presented. The ITS1 region of the ribosomal DNA was used to design species-specific primers. The specificity of the primers together with the melting curve profile provided a reliable qualitative identification and discrimination between the two Karlodinium species. Additionally, a simple and rapid DNA extraction method was used. Standard curves were constructed from 10-fold dilutions of cultured microalgae cells. Finally, the applicability of the assay was tested with field samples collected from Alfacs Bay. Results showed a significant correlation between qPCR determinations and light microscopy counts (y = 2.838 x + 564; R2 = 0.936). Overall, the qPCR method developed herein is specific, rapid, accurate, and promising for the detection of these two Karlodinium species in environmental samples.
KeywordsKarlodinium veneficum Karlodinium armiger Dinoflagellate Quantitative PCR ITS rDNA DNA extraction
The authors would like to thank Núria Orra and Rosa Fibla for the initial development of the qPCR methodology as well as María Rey for field sample counting by optical microscopy and her help on culturing of microalgae, José Luis Costa for collecting Karlodinium samples at Alfacs Bay, and Josep Fumadó for kindly helping to test the environmental samples by qPCR.
This study received financial support from the Ministerio de Economía, Industria y Competitividad through the SEASENSING (BIO2014-56024-C2-2-R) and PURGA DE MAR (IPT-2011-1707-310000) projects. This study also received support from CERCA Programme/Generalitat de Catalunya. Anna Toldrà received her PhD grant (2015PMF-PIPF-67) from IRTA-Universitat Rovira i Virgili-Banco Santander.
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