Development and comparison of loop mediated isothermal amplification assay and polymerase chain reaction based on major capsid protein gene for detection of CyHV-2 infection in goldfish Carassius auratus (L.)

Abstract

Cyprinid herpesvirus 2 (CyHV-2) is the etiological agent of goldfish herpesviral haematopoietic necrosis (GHVHN) that caused high economic losses in goldfish aquaculture. In this study, a loop-mediated isothermal amplification (LAMP) assay as well as a polymerase chain reaction (PCR) targeting major capsid protein (MCP) gene of CyHV-2 were standardised and evaluated for detection of CyHV-2. CyHV-2 was purified from infected fantail goldfish fin (FtGF) cells using ultracentrifugation and used as template for developing the diagnostic assays. The new LAMP and PCR assays are highly specific and did not amplify the nucleic acids of other fish pathogens tested, namely spring viraemia of carp virus (SVCV), Cyprinid herpesvirus-3 (CyHV-3), and viral nervous necrosis virus (VNNV), Aeromonas hydrophila, A. veronii, A. caviae, Edwardsiella tarda, Vibrio anguillarum, V. parahaemolyticus, V. harveyi and Proteus hauseri. Among the two assays developed, LAMP was found to be more sensitive, capable of detecting 10 copies of the plasmid construct containing 942 bp fragment of MCP gene of CyHV-2, while PCR could detect only 100 copies. The LAMP assay developed is a simple, rapid and reliable method for detection of CyHV-2 infection which can also be used in field conditions. Keywords: Cyprinid herpesvirus-2 (CyHV-2), Diagnostic assays, Goldfish, LAMP assay, MCP gene, PCR