Abstract: In order to screen one or more applicably universal primers for environmental DNA(eDNA) research based on high-throughout sequencing technology, five pairs of primers of fish mitochondrial genome fragment were chosen from literatures, which could amplify the partial sequences of Cyt b gene, 16S rDNA and COⅠ gene. After initial amplification on two water eDNA samples, it was found that CY45M and 1634M had better amplification effects and their products could be applied to the subsequent high-throughout sequencing. Meanwhile, no purpose band could be amplified using the other three primers, including CYFM, 16ACM and COⅠM. After the next amplification on 7 water eDNA samples using CY45M and 1634M, it was found that all the eDNA were amplified by these two pairs of primers, and the PCR products presented clear bright bands by gel electrophoresis. According to the high-throughout sequencing and BLAST results, the sequences gained by both of CY45M and 1634M could be annotated on 11 fish species.Nevertheless, there were dissimilarity and repeatability upon these fish species. In summary, the CY45M and 1634M were both suitable universal primers for study on eDNA of fish community structure in Qiantang River.

Key words: primer verification, environmental DNA, high throughput sequencing, Qiantang River