A fast and accurate method for specific detection and quantification of the bloom-forming microalgae Karlodinium veneficum in the marine environment

Abstract

Karlodinium veneficum is a toxic benthic globally distributed dinoflagellate which has direct impacts on human health and the environment. Early and accurate detection of this harmful algal bloom-forming species could be useful for potential risks monitoring and management. In the present work, a real-time PCR targeting the internal transcribed spacer ribosomal DNA region for the specific detection and absolute quantification of K. veneficum was designed. Then, the assay conditions were adjusted and validated. The developed qPCR was highly specific for the target species and displayed no cross-reactivity with closely related dinoflagellates and/or other microalgal species commonly distributed along the Tunisian coast. Its lowest detection limit was 5 rDNA copies per reaction, which is often considered satisfying. qPCR assay enumeration accuracy was evaluated using artificially inoculated environmental samples. The comparison of the cell abundance estimates obtained by qPCR assay with the theoretical estimates showed no statistically significant difference across a range of concentrations. We suggest that the qPCR approach developed in the present study may be a valuable tool to investigate the distribution and seasonal dynamics of K. veneficum in marine environments.

Keywords: Absolute quantification; Bivalve mollusks; Internal transcribed spacer; Karlodinium veneficum; Toxic microalgae; qPCR assay.