Application Of PCR-DGGE Assay And Real-Time PCR Assay For Detection Of Harmful Algae

Red tide is an ecologic phenomenon that seawater turns red, brown, or yellow, when oceanic plankton overly propagate or congregate. Red tide, especially harmful algae blooms have negatively impacted the aquaculture, fisheries and tourism industries as well as the environment and public health. To understand red tide and further control and prevent the occurring of HABs, it is foremost and essential to ascertain the causative species, while the efficient detection methods should be developed to accelerate the identification and quantification aiming at monitoring the composition of organisms in the natural water. In this study, Symbiodinium sp. Freudenthal, Heterosigma akashiwo Hada, and Prorocentrum micans Ehrenberg were used to establish a method combining PCR-DGGE and Real-Time PCR to identify and quantify harmful algae.For identification, universal gene primers for PCR based on18S rDNA from these algae were designed, and then corresponding DNA fragments were PCR amplified and DGGE analysis was conducted. Fortunately, the differences in the electrophoresis pattern suggested that these three algae could be separated by DGGE.For quantification, species-specific primers for every alga were designed based on ITS rDNA; the primers’specificity was confirmed by BLAST search and then tested by PCR amplification of other algae species in our laboratory. Next, the standard curve was constructed using the Ct value and the cultured algal cells number. A test using a simulated field seawater sample showed that cell enumeration results obtained by Real-Time PCR agreed well with those obtained by cell counting under a microscope.We also determined that algal cells or crude cell lysates could be used as the template for PCR directly, saving test time and potentially making quantification more accurate due to less DNA loss.