Multiplex PCR methods for the species-specific detection and quantification of Prymnesium parvum (Haptophyta)

Multiplex polymerase chain reaction (PCR) assays were developed for detecting and quantifying Prymnesium parvum wherein suites of primers simultaneously amplify four species- and gene-specific products using genomic DNA or whole cells for template. With conventional PCR, amplification products were easily resolved by gel electrophoresis, generating a diagnostic banding pattern. Gene-specific fluorescent molecular beacons were designed for use with real-time quantitative PCR (qPCR). Both methods were capable of detecting as few as one or two cells in 50 cycles. The species and gene specificities of the assays were evaluated using isolates (and mixtures) of P. parvum, related species, and out-groups. Cell counts using qPCR to evaluate environmental samples were comparable to mean values obtained from manual counts and had lower standard deviations. This presents a significant improvement in DNA-based detection technology, enhanced by the rapid and simultaneous confirmation of four species-specific products and the ability to detect several widely separated geographic isolates of P. parvum.