Development of a real-time PCR for rapid detection of Skeletonema costatum

Sequences of the rDNA and ITS deduced from Skeletonema costatum were used for designing species-specific primers, and a real-time fluorescent quantitative polymerase chain reaction (RFQ-PCR) method was developed for quantitative detection of S. costatum. Seven sets of primers and probes were designed, and were applied to specificity check with PCR and dot-blot analysis. Probe 6 and primer 6 (F/R) were selected to develop a RFQ-PCR method for S. costatum. A calibration curve was constructed with Cycle Threshold value against visual counted cell number. The value of the curve was tested with other S. costatum samples, which were assayed with both RFQ-PCR method and visual count under microscope.