Quick hassle-free detection of cyprinid herpesvirus 2 (CyHV-2) in goldfish using recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay

Cyprinid herpesvirus 2 (CyHV-2) is the etiological agent of herpesviral hematopoietic necrosis disease (HVHND), which causes severe mortality in ornamental goldfish (Carassius auratus), crucian carp (Carassius auratus), and gibel/prussian carp (Carassius gibelio). Quick and hassle-free point-of-care detection of CyHV-2 is vital for the maintenance of ornamental fish health. In this manuscript, we describe the development of a rapid and sensitive RPA (recombinase polymerase amplification) assay, coupled with lateral flow dipsticks (LFD), that can achieve sensitive diagnosis of CyHV-2 in goldfish within 20 min at 36 °C with the satisfactory detection limit of 102 gene copies per reaction. This is the first report wherein major capsid protein (MCP) of CyHV-2 was targeted for RPA-LFD assay development. The assay did not show any cross-reactivity with other viral pathogens like cyprinid herpesvirus 3 (CyHV-3), spring viremia of carp virus (SVCV), infectious spleen and kidney necrosis virus (ISKNV), and viral nervous necrosis virus (VNNV). Furthermore, screening of CyHV-2 infection in CyHV-2-infected goldfish did not yield any false positive/negative results. In short, the RPA-LFD assay developed in this study presents a simple, rapid, and sensitive method for point-of-care diagnosis of CyHV-2, especially under resource-limited conditions.