Detection and quantification of the red tide dinoflagellate Karenia brevis by real-time nucleic acid sequence-based amplification

Abstract

Nucleic acid sequence-based amplification (NASBA) is an isothermal method of RNA amplification that has been previously used in clinical diagnostic testing. A real-time NASBA assay has been developed for the detection of rbcL mRNA from the red tide dinoflagellate Karenia brevis. This assay is sensitive to one K. brevis cell and 1.0 fg of in vitro transcript, with occasional detection of lower concentrations of transcript. The assay did not detect rbcL mRNA from a wide range of nontarget organisms and environmental clones, while 10 strains (all tested) of K. brevis were detected. By the use of standard curves based on time to positivity, concentrations of K. brevis in environmental samples were predicted by NASBA and classified into different levels of blooms per the Florida Fish and Wildlife Conservation Commission (FWC) system. NASBA classification matched FWC classification (based on cell counts) 72% of the time. Those samples that did not match were off by only one class. NASBA is sensitive, rapid, and effective and may be used as an additional or alternative method to detect and quantify K. brevis in the marine environment.

FIG. 1.

Typical amplification plot of K. brevis cell standards (squares, 1,000 cells; circles, 100 cells; triangles, 10 cells; diamonds, 1 cell; ×, no cells). Samples were run in triplicate for each concentration. The horizontal line on the figure indicates the threshold fluorescence used to calculate the TTP value. ΔRn, relative fluorescence.

FIG. 2.

Typical standard curve generated from K. brevis cells (r² = 0.92). Points at each concentration represent a single RNA extraction, run in triplicate NASBA reactions. Ct, threshold cycle.