STUDY ON TOTAL DNA EXTRACTION METHODS OF SAPROLEGNIA

Saprolegnia are parasitic on fish and their eggs and known as the most important pathogens in freshwater fish. Infec2tious diseases caused by water molds can cause losses of freshwater fish in both nature and commercial fish farms. It is very im2portant to obtain the high quality and effective total DNA to study the mechanism of molecular nosogenesis, identify the speciesusing molecular marker and gene diagnoses.In this study, Saprolegnia parasitica, S. ferax, S. dolica, S. sp1and S. spwere used as test materials. The DNA extrac2tion methods of Lysozyme, CTAB, improved CTAB, CH4N2O and SDSwere compared. Firstly, the 30 mg (net weight) of freshlysubcultured mycelium of five different pathogenetic fish fungi were taken to freeze at - 70 e for 30min in 1. 5mL Eppendorftubes, respectively. Then they were transferred to room temperature and grinded using appropriate abrasive tool. This processwas repeated once. Secondly, the trituration of five mycelium were digested by lysozyme digestion, CTAB digestion, improvedCTAB digestion, CH4N2O digestion and SDS digestion, respectively, then extracted DNA using relevant processes. The yield andquality of DNA were evaluated with ultraviolet radiation spectrophotometer and PCR of the ITS rDNA. The result of ultraviolet ra2diation spectrophotometer showed that DNA can be obtained using five different methods and the genomic DNA extracted with im2proved CTABmethod had the best quality and yield, the A260/ A280 ratio was between 1. 79) 1. 82 and the concentrat ion ofDNAwas about 45Lg/mL. The result of PCR showed that fiveDNA samples extracted with improved CTABmethod can bewell ampli2fied and obtained the bright, no tail and trim electrophoresis strip. However, there were unclear or vacant electrophoresis stripsin the DNA samples extracted with other methods. Therefore, the DNA extraction method of improved CTABwas the best methodto gain the DNA of Saprolegnia for the study of molecular level.