Development of real-time PCR assays for rapid detection of Pfiesteria piscicida and related dinoflagellates

Pfiesteria complex species are heterotrophic and mixotrophic dinoflagellate s that have been recognized as harmful algal bloom species associated with adverse fish and human health effects along the East Coast of North America , particularly in its largest (Chesapeake Bay in Maryland) and second large st (Albermarle-Pamlico Sound in North Carolina) estuaries. In response to i mpacts on human health and the economy, monitoring programs to detect the o rganism have been implemented in affected areas. However, until recently, s pecific identification of the two toxic species known thus far, Pfiesteria piscicida and P. shumwayae (sp. nov.), required scanning electron microscop y (SEM). SEM is a labor-intensive process in which a small number of cells can be analyzed, posing limitations when the method is applied to environme ntal estuarine water samples. To overcome these problems, we developed a re al-time PCR-based assay that permits rapid and specific identification of t hese organisms in culture and heterogeneous environmental water samples. Va rious factors likely to be encountered when assessing environmental samples were addressed, and assay specificity was validated through screening of a comprehensive panel of cultures, including the two recognized Pfiesteria s pecies, morphologically similar species, and a wide range of other estuarin e dinoflagellates. Assay sensitivity and sample stability were established for both unpreserved and fixative (acidic Lugol's solution)-preserved sampl es. The effects of background DNA on organism detection and enumeration wer e also explored, and based on these results, we conclude that the assay may be utilized to derive quantitative data. This real-time PCR-based method w ill be useful for many other applications, including adaptation for field-b ased technology.