Detection and quantification of Pseudo-nitzschia australis in cultured and natural populations using LSU rRNA-targeted probes

Pseudo-nitzschia australia Frenguelli is a marine pennate diatom associated 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 time consuming because of a requirement for scanning electron microscopy. Here, we apply large-subunit ribosomal RNA (LSU rRNA)-targeted oligonucleotides in whole-cell and sandwich hybridization formats to identify and enumerate this species collected from pure cultures and natural populations. Whole-cell hybridization employed fluorescently labeled probes, filter-based sample processing, and epifluorescence microscopy to enumerate labeled cells. In contrast, sandwich hybridization was accomplished by homogenizing cells in a chaotropic solution and performing two hybridization reactions: capture of LSU rRNA using an oligonucleotide 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 proportional to the abundance of target species in the original sample. The sandwich hybridization assay was semiautomated with a robotic processor. Both whole-cell and sandwich hybridization are useful techniques for identifying 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.