Detection and quantification of Prymnesium parvum (Haptophyceae) by real-time PCR

Abstract

The ichthyotoxic species Prymnesium parvum (Haptophyceae) is difficult to quantify in a microscopy-based monitoring programme, because the cells are very small, fragile and their morphology can be distorted by the use of fixatives. In the attempt to overcome these problems, a real-time PCR-based method for the rapid and sensitive identification and quantification of P. parvum was developed. A quantitative real-time PCR assay was optimized with primers designed on the internal transcribed spacer 2 rDNA region of P. parvum. This PCR assay was specific, showing no amplification of DNA extracted from closely related species, and sensitive. Moreover, this method was able to detect and reliably quantify P. parvum cells in preserved environmental samples artificially spiked with known amounts of cultured cells. Considering the specificity, sensitivity and applicability to preserved environmental samples, this method may be a useful tool for the monitoring of this toxic species. The real-time PCR method described in this study may represent a progress towards the rapid detection and quantification of P. parvum cells in water-monitoring programmes, allowing the early application of strategies to control bloom events, such as the use of clay minerals.