Development of a SYT09 based real-time PCR probe for detection and quantification of toxic dinoflagellate Karlodinium veneficum (Dinophyceae) in environmental samples

摘要

Conventional methods to identify the fish-killing dinoflagellate Karlodinium veneficum rely on rDNA sequence and scanning electron microscopy analyses that are not suitable for processing a large number of samples. To overcome this difficulty, SYTO9 and TaqMan format real-time PCR probes were developed for the internal transcribed spacer rDNA region for rapid detection and quantification of K. veneficum in the marine environment. Assay specificity, sensitivity, and accuracy were confirmed by testing against related organisms, sequencing PCR amplicons from field samples, and comparing real-time PCR results with microscopic counts. The SYTO9-based assay produced highly reproducible DNA melting curves over a broad range of starting DNA template. When the SYTO9- and TAqMan-based assays were comapred in the quantitative measurements, they showed comparable results and reasonable reproducibility. The K. veneficum-specific assays were used in conjunction with Pseudopfiesteria shumwayae (Dinophyceae)-, Pfiesteria piscicida (Dinophyceae)-, and Cryptoperidiniopsis brodyi (Dinophyceae)-specific real-time PCR probes to investigate temporal changes in abundances of these four species in Chinhae Bay, South Korea. The field survey revealed that K. veneficum was dominant (maximum 970 cells ml(-1)) among those species: whereas, Pfiesteria species rarely occurred. These findings suggest that the SYTO9- and TAqMan-based assays are specific and sensitive for detecing and quantifying K. veneficum in the environment. The wide distribution of K. veneficum also indicates potential fish kills by this organism in Chinhae Bay.