Dual PCR Detection of Type 2 and 3 Cyprinus Herpesvirus

  Objective  A PCR method for simultaneously detecting Type 2 and Type 3 Cyprinid herpesvirus that cause serious diseases on Cyprinidae was developed and tested for clinical diagnosis.

  Methods   Two pairs of specific primers were designed according to the conserved sequences of the DNA polymerase gene of the two types of virus, CyHV-2 and CyHV-3. PCR reaction conditions of the method were optimized. The assay was applied on the stored tissue samples of diseased Carassius auratus and Cyprinus carpio to verify validity of the methodology.

  Result  The newly developed Dual PCR Assay amplified specific bands with the base numbers of 715 bp for CyHV-2 and 456 bp for CyHV-3. It exhibited high sensitivity with a detection limit of 100 copies·μL⁻¹. On the 18 clinical samples, 3 were found to be CyHV-2 and 3 CyHV-3 with a positive detection rate of 33.3%. Furthermore, the assay successfully identified the two type viruses in a mixed sample of CyHV-2 and CyHV-3 in a challenge test.

  Conclusion   The Dual PCR Assay demonstrated high specificity and sensitivity in simultaneously detecting CyHV-2 and CyHV-3. It could be adequately applied for rapid diagnosis of the viral diseases on carps.