International accreditation of sandwich hybridisation assay format DNA probes for micro-algae

The sandwich hybridisation assay (SHA) is a DNA probe-based method for rapid identification and enumeration of toxic micro-algae which uses species specific oligonucleotide probes targeted at ribosomal RNA. It is suited to fragile micro-algal cells which commonly collapse during the fixation stage of sample collection, compromising identification by traditional microscopy. The assay has been available for research for several years, but was validated and accepted for international accreditation for commercial laboratory use in New Zealand in May 2004 (International Accreditation New Zealand: ISO 17025). During the validation of the raphidophyte assay, some discrepancies were noted between SHA cell concentration estimates and traditional light microscope cell counts. Higher SHA estimates were recorded when blooms had collapsed but rRNA was still present in sea water. Conversely, higher traditional cell counts occurred when sample delivery was delayed more than 48 h, presumably owing to degradation of rRNA in the live cultures used for the SHA. SHA cell concentration estimates of the toxic diatom bloom-former Pseudo-nitzschia australis were also compared with whole cell format DNA probe counts and traditional microscope counts; SHA counts were comparable for the three methods tested.