Initial assessment of triplex PCR assay application for detection of toxic dinoflagellates, Alexandrium species (Dinophyceae), in environmental samples

Includes bibliographical references (leaves. 23-28).The frequency and geographical range of hannful algal blooms (HABs) are believed to be on the increase, with adverse affects on marine and human health making the implementation of stringent controls governmg monitoring programmes commonplace. The South African monitoring programme was established in 1989 and relies upon microscopic identification of HAB species. Microscopic identification is labour-intensive, requiring a high level of taxonomic expertise, and could be considered impractical for routine monitoring where analysis of large numbers of samples is required. Novel monitoring techniques, focusing mainly on probe technology, are being developed for rapid, unequivocal identification and enumeration of HAB species. In this study, a triplex peR assay, incorporating a genus-specific ribosomal DNA primer designed from phylogenetic studies on local Alexandrium populations, was optimised for application to environmental samples and tested against natural assemblages containing Alexandrium minutum. Specific positive results were consistently generated for samples containing A. minutum. Samples absent of A. minuturn cells did not generate the Alexandrium-specific amplicon. The absolute detection limit of 440 A. minutum cells r1 for this assay was established. Effects of non-target cells on the sensitivity of the assay were also investigated: although a decrease in sensitivity was found, A. minutum cells could still be detected in the presence of 100 times more non-target cells. This assay has been shown to be a useful tool for unequivocal identification of A. minutum cells within local environmental samples