An advanced tool, droplet digital PCR (ddPCR), for absolute quantification of the red-tide dinoflagellate, Cochlodinium polykrikoides Margalef (Dinophyceae)

Abstract

To quantify the abundance of the harmful marine dinoflagellate Cochlodinium polykrikoides in natural seawaters, we developed an innovative procedure based on the droplet digital PCR (ddPCR) with C. polykrikoides-specific primers targeting the internal transcribed spacer (ITS). The abundance of C. polykrikoides was determined through the specific copy number of target DNA per cell in both culture and field samples. The average copy number per C. polykrikoides cell as determined via the ddPCR was 157 with using known cell numbers and a simplified protocol for preparing DNAs. C. polykrikoides cell numbers, in samples collected from different locations, calculated via ddPCR were in agreement with direct cell counts using a microscope. The ddPCR protocol was then used to measure the abundance of C. polykrikoides in nearshore and offshore waters of the southern coast of Korea. Practical application of this method demonstrated its usefulness in reducing time spent on analysis and for increasing estimate accuracy.