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

摘要

The detection and quantitative analysis of Pythium porphyrae zoospores was performed by PCR using PP-1 and PP-2 primers specific to the internal transcribed spacer region of P. porphyrae. To estimate the amount of fungal zoospores of P. porphyrae, an internal standard plasmid (pPPISC) containing a modified DNA fragment was constructed. Both ends of this fragment were complementary to the PCR primers. Amplification using primers PP-1 and PP-2 produced DNA fragments of approximately 700 and 400 bp from the target DNA of P. porphyrae zoospores and from the pPPISC, respectively. To perform quantitative PCR, known quantities of pPPISC were added to reaction mixtures containing the experimental DNAs extracted from zoospores. After a co-amplification reaction, the two different sized PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. The number 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 competitor pPPISC are useful tools for the quantitative analysis of P. porphyrae zoospores in seawater from Porphyra cultivation farms.