Detection and quantitative analysis of zoospores of Pythium porphyrae, causative organism of red rot disease in Porphyra, by competitive PCR

The detection and quantitative analysis of Pythium porphyrae zoospores was performed by PCR using PP-1 and PP-2 primers specific to the internal trans cribed spacer region of P. porphyrae. To estimate the amount of fungal zoos pores of P. porphyrae, an internal standard plasmid (pPPISC) containing a m odified DNA fragment was constructed. Both ends of this fragment were compl ementary to the PCR primers. Amplification using primers PP-1 and PP-2 prod uced DNA fragments of approximately 700 and 400 bp from the target DNA of P . porphyrae zoospores and from the pPPISC, respectively. To perform quantit ative PCR, known quantities of pPPISC were added to reaction mixtures conta ining the experimental DNAs extracted from zoospores. After a co-amplificat ion reaction, the two different sized PCR products were separated by agaros e gel electrophoresis and visualized by ethidium bromide staining. The numb er of zoospores was estimated by comparing the fluorescence intensities of the PCR products using a charge-coupled device image analyzer. The results show that competitive PCR using P. porphyrae specific primers and competito r pPPISC are useful tools for the quantitative analysis of P. porphyrae zoo spores in seawater from Porphyra cultivation farms.