A real-time recombinase polymerase amplification assay for the rapid detection of CyHV2

Abstract:

[Background] Herpes viral haematopoietic necrosis (HVHN) caused by cyprinid herpesvirus 2 (CyHV2) is one of the main diseases in crucian carp breeding, causing serious economic losses. The disease has no effective therapeutic treatment, therefore early detection of the virus and efficient control is an effective way to prevent its outbreak. [Objective] A real-time recombinase polymerase ampli?cation (RPA) assay for CyHV2 orf72 gene was established, and its specificity and sensitivity were evaluated. [Methods] Specific primers and probes were designed in the conserved region by comparing the orf72 nucleotide sequences between the five CyHV2 strains. Five reaction temperatures were set to optimize the conditions of real-time RPA assay. Under the optimal conditions, the specificity of the real-time RPA assay was verified among different species. The sensitivity of real-time RPA and qPCR was compared using gradient diluted CyHV2 positive DNA as template. [Results] Real-time RPA assay can quickly and accurately detect CyHV2 virus within 20 minutes at 37.8 °C, with high specificity, no cross-reaction with other viruses, and the same sensitivity as qPCR. [Conclusion] The real-time RPA assay developed in this study can be used for rapid on-site detection of CyHV2.