Molecular detection of the brevetoxin‐producing dinoflagellate Karenia brevis and closely related species using rRNA‐targeted probes and a semiautomated sandwich hybridization assay1

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Abstract
Brevetoxins produced by the marine dinoflagellate Karenia brevis (C. C. Davis) G. Hansen et Moestrup cause neurotoxic shellfish poisoning (NSP) in human consumers and also endanger a variety of coastal wildlife. In the eastern Gulf of Mexico the presence and abundance of this species have traditionally been monitored using light microscopy (LM) observations of whole water samples. Various molecular probe methods now enable detection of multiple species from a single sample, allowing rapid sample analysis. We describe the development of sandwich hybridization assays (SHAs) for Karenia brevis, K. selliformis Haywood, Steid. et L. MacK., K. mikimotoi (Miyake et Kominami ex M. Oda) G. Hansen et Moestrup, K. papilionacea Haywood et Steid., the Karlotoxin‐producer Karlodinium veneficum (D. Ballant.) J. Larsen (=K. micrum), and Gymnodinium aureolum (Hulburt) G. Hansen, comb. nov. The assays require no nucleic acid purification and use LSU rRNA‐targeted probes and a semiautomated, 96‐well plate format. Probes tested in matrix format were specific relative to rRNAs of all nontarget species used. The response of the SHA for a constant number of K. brevis cells per unit volume of homogenate depended on the growth status of a culture, decreasing for senescent cells relative to actively growing cells. The results of preliminary field tests of the K. brevis SHA indicated that cells collected from natural populations tended to return a lower signal than those harvested from laboratory cultures, but these results are nonetheless very encouraging. These preliminary field studies show that robust standards are required for cell identification and enumeration, with which new methods can be compared.