The use of quantitative polymerase chain reaction for the detection and enumeration of the harmful alga Aureococcus anophagefferens in environmental samples along the United States East Coast

Linda C. Popels, S. Craig Cary, David A. Hutchins, Rachel Forbes, Frances Pustizzi, Christopher J. Gobler,Kathryn J. Coyne

Limnol. Oceanogr. Methods 1:92-102 (2003) | DOI: 10.4319/lom.2003.1.92

ABSTRACT: Aureococcus anophagefferens is a small pelagophyte that has caused harmful brown tide blooms in New York, New Jersey, Rhode Island, and Maryland. It is visually indistinguishable from other small algae in environmental samples using standard light microscopy, and current species-specific immunofluorescent counting techniques are not sensitive enough to detect concentrations lower than approximately 5000 cells mL–1. A TaqMan molecular probe was developed that is specific for A. anophagefferens based on a unique 18S rDNA sequence to detect cells at low levels. Real-time quantitative PCR (qPCR) on an ABI Prism 7700 Sequence Detection System was used to generate a standard curve of cycle thresholds (Ct) with known concentrations of A. anophagefferens (R2 = 0.98). Cell concentrations of A. anophagefferens in environmental samples are determined by linear regression analysis, with a sensitivity of approximately 1 cell mL–1. Estuarine and coastal water samples were collected along the U.S. East Coast from Florida to Delaware, and during a transect from the Delaware River to offshore mid-Atlantic waters, then analyzed using qPCR. Aureococcus has a wide distribution at background concentrations as far south as Northern Florida, far outside the presently recognized range of the organism. It was also found in offshore Atlantic samples, suggesting a possible oceanic source for bloom inoculation. qPCR allows for detection of cells at prebloom levels, which is essential for effective management and prediction of brown tide blooms.