Molecular detection and quantitation of the red tide dinoflagellate Karenia brevis in the marine environment

Abstract

A real-time reverse transcription-PCR method targeting the rbcL gene was developed for the detection and quantitation of the Florida red tide organism, Karenia brevis. The assay was sensitive to less than 1 cell per reaction, did not detect rbcL from 38 nontarget taxa, and accurately quantitated K. brevis organisms in red tide samples from around Florida. These studies have resulted in a sensitive and specific method for K. brevis detection in the marine environment.

FIG. 1.

Neighbor-joining phylogenetic tree based on deduced amino acid sequences with a Poisson distance correction showing relationships between form I rbcL sequences from K. brevis and other phytoplankton species, as well as clones obtained on a cruise to the Mississippi River plume in the Gulf of Mexico. Boldface taxa were tested by real-time RT-PCR as nontarget controls. There were many taxa tested as nontarget strains whose rbcL sequences were not available in GenBank, and closest sequenced representatives are underlined.

FIG. 2.

Real-time RT-PCR standard curve generated from the APC6 clone 15 transcript showing the linearity of the method, covering 7 orders of magnitude (filled circles [trendline]). Also shown are amplification results from K. brevis cellular extracts corresponding to 100 cells, 10 cells, and 1 cell per reaction (open circles).

FIG. 3.

Comparison of microscopy cell counts and real-time RT-PCR-inferred cell counts from natural bloom samples.