Real-time polymerase chain reaction assays for rapid detection and quantification of Noctiluca scintillans zoospore

Abstract

Application and availability of real-time polymerase chain reaction (PCR) assay to detect and quantify the Noctiluca scintillans zoospore were investigated seasonally. Specific primer set for N. scintillans 18S rDNA was designed and applied to real-time PCR assay using the serial dilutions of N. scintillans zoospores. The real-time PCR assays with Ns63F and Ns260R primers were applied to sea water samples collected weekly in Manazuru Port of Sagami Bay, Japan from April 2005 to June 2006. We developed effective DNA preparation steps for collecting the template DNA of N. scintillans zoospore: size fraction and filter concentration of the water samples, fixation with Lugol solution, cell lysis, and purification. This method is useful for the monitoring of the zoospores of N. scintillans, and can also be used for other small and physiologically fragile planktonic cell. Variation in the density of zoospore was successfully detected in the field samples. The peak density of N. scintillans zoospore was observed to occur just before or at the same time as the peak of the vegetative cells. Moreover, zoospores were detected in seawater even when the vegetative cells were not observed. The presence of zoospore was found all year round in the present study. In this regards, this information is essential for the study of the life cycle and seasonal variation of N. scintillans in the coastal waters.