Rapid and accurate identification by real-time PCR of biotoxin-producing dinoflagellates from the family gymnodiniaceae

Abstract

The identification of toxin-producing dinoflagellates for monitoring programmes and bio-compound discovery requires considerable taxonomic expertise. It can also be difficult to morphologically differentiate toxic and non-toxic species or strains. Various molecular methods have been used for dinoflagellate identification and detection, and this study describes the development of eight real-time polymerase chain reaction (PCR) assays targeting the large subunit ribosomal RNA (LSU rRNA) gene of species from the genera Gymnodinium, Karenia, Karlodinium, and Takayama. Assays proved to be highly specific and sensitive, and the assay for G. catenatum was further developed for quantification in response to a bloom in Manukau Harbour, New Zealand. The assay estimated cell densities from environmental samples as low as 0.07 cells per PCR reaction, which equated to three cells per litre. This assay not only enabled conclusive species identification but also detected the presence of cells below the limit of detection for light microscopy. This study demonstrates the usefulness of real-time PCR as a sensitive and rapid molecular technique for the detection and quantification of micro-algae from environmental samples.

Figure 1

Mean A260/A280 ratios and cycle threshold (Ct) values for replicate DNA extractions of Karenia mikimotoi. Error bars are ± standard error of three replicate DNA extractions.

Figure 2

Gymnodinium catenatum cell number estimates by real-time PCR and light microscopy (LM) from natural phytoplankton samples spiked with cultured G. catenatum cells. Error bars are ±standard error from triplicate LM analyses and real-time PCR assays.

Figure 3

Gymnodinium catenatum cell number estimates by real-time PCR and light microscopy (LM) from samples collected at Manukau Bay, New Zealand. Error bars are ±standard error from replicate real-time PCR assays.