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 signal probe that bound more conserved regions of the molecule. The signal probe served as a platform for an enzyme-driven colourimetric reaction; presence of P. australis in the sample resulted in a macroscopic colour change of the solid support.Both whole cell and sandwich hybridization methods are useful techniques for discriminating P. australis from its closely related congeners. The relative advantages and disadvantages of each technique are discussed. With respect to future applications, choice of one method over another will likely depend on the particular question at hand and the type of data one wishes to obtain. At present, sandwich hybridization appears faster, easier, and more amenable to automation. For these reasons, it may be a more appropriate technique for routine, rapid enumeration of potentially toxic microalgal species in large numbers of environmental samples.