DETECTION AND QUANTIFICATION OF PSEUDO-NITZSCHIA AUSTRALIS IN CULTURED AND NATURAL-POPULATIONS USING LSU RIBOSOMAL-RNA-TARGETED PROBES

Pseudo-nitzschia australia Frenguelli is a marine pennate diatom assoc iated with the production of domoic acid-a neuroexcitatory amino acid linked to illness and mortality of humans and wildlife. Distinguishing P. australia from its co-occurring congeners is labor intensive and t ime consuming because of a requirement for scanning electron microscop y. Here, we apply large-subunit ribosomal RNA (LSU rRNA)-targeted olig onucleotides in whole-cell and sandwich hybridization formats to ident ify and enumerate this species collected from pure cultures and natura l populations. Whole-cell hybridization employed fluorescently labeled probes, filter-based sample processing, and epifluorescence microscop y to enumerate labeled cells. In contrast, sandwich hybridization was accomplished by homogenizing cells in a chaotropic solution and perfor ming two hybridization reactions: capture of LSU rRNA using an oligonu cleotide coupled to a macroscopic solid support and binding of signal probe to a region of LSU rRNA near that of the capture site. Sandwich hybrids were detected colorimetrically; color intensity was proportion al to the abundance of target species in the original sample. The sand wich hybridization assay was semiautomated with a robotic processor. B oth whole-cell and sandwich hybridization are useful techniques for id entifying P. australia as it occurs in nature. Sandwich hybridization potentially offers the most rapid and simple means to accomplish this task when screening large numbers of environmental samples.