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 (Fig. 1) The assay did not detect rbcL mRNA from a wide range of non-target organisms and environmental clones while ten strains (all tested) of K. brevis were detected (Table 1). Using standard curves based on time to positivity (TTP), concentrations of K. brevis in environmental samples were predicted by NASBA and classified into different levels of blooms as per the Florida Fish and Wildlife Conservation Commission (FWC). NASBA classification matched FWC classification (based on cell counts) 72% of the time (Table 2). 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.
Reference: Erica T. Casper, John H. Paul, Matthew C. Smith, and Michael Gray. 2004. The detection and quantification of the red tide Dinoflagellate Karenia brevis by real-time NABSA. Appl. Environ. Microbiol. 70:4727-4732.
Karenia brevis is the Florida Red Tide organism capable of causing massive fish and marine mammal mortalities, the contamination of shellfish, and respiratory distress in animals and humans.
Table 1. Specificity of the K. brevis NASBA assay. Click here to view Table 1.
Table 2. Comparison of K. brevis cell counts with NASBA determined counts for bloom and non-bloom samples around Florida. Letters in parenthesis are the level designation (N= none, < 1000 cells/L; VL = very low, 1000-10,000 cells/L; L = low, 10,000-100,000 cells/L; M= medium, 100,000 to 106/L; H = high, > 106/L as modified from the Florida Wildlife Commission levels (http://www.floridamarine.org/features/view_article.asp?id=9670).
Click here to view Table 2.