Abstract: To develop one method based on pyrosequencing for high throughput, automated and rapid detection of Cyprinid herpesvirus 3 (CyHV-3), specific primer pairs for amplification and sequencing were designed that targeting conserved region of ORF55, ORF79 and ORF92 gene of CyHV-3, and the optimal primers were screened by PCR amplification. The results of sensitivity and specifity test showed the primers against ORF79 gene are the best primers for PCR amplification with the detection limit as least as 100 copies and specific for the detection of CyHV-3. The sequencing result of pyrosequencing using PCR product indicated the obtained sequence shares 100% identity with CyHV-3-U, I, J and GZ11 isolates. Therefore, this assay could be used as a new tool for the diagnosis of CyHV-3 infections on a large scale samples in ports of exit and entry due to its convenient and rapid characteristic.

Key words: Cyprinid herpesvirus 3 (CyHV-3), koi herpesvirus (KHV), pyrosequencing, detection