Identification of Pseudo-nitzschia australis (Bacillariophyceae) using rRNA-targeted probes in whole cell and sandwich hybridization formats

Abstract

Large-subunit ribosomal RNA (LSU rRNA)-targeted oligonucleotides specific for Pseudo-nitzschia australis Frenguelli were used to discriminate that species from cultured isolates of P. pungens (Grunow) Hasle, P. multiseries (Hasle) Hasle, P. delicatissima (P.T. Cleve) Heiden, P. pseudodelicatissima (Hasle) Hasle, P. fraudulenta (P.T.Cleve) Heiden, P. heimii Manguin, and P. americana (Hasle) Fryxell using whole cell (in situ) and sandwich hybridization. Whole cell hybridization involved application of fluorescently labelled probes to chemically preserved (intact) cells. Cells that retained the probe were visualized using epifluorescence microscopy. In contrast, sandwich hybridization was initiated by homogenizing live cells to liberate their contents. Detection of P. australis was accomplished by capturing LSU rRNA from unpurified cell lysates using a species-specific oligonucleotide attached to a solid support. Captured molecules were washed free of other lysate and hybridized to a biotinylated si...