Development of quantitative polymerase chain reaction assay for detection of scale drop disease virus (SDDV) in Asian sea bass

Abstract

Scale drop disease virus (SDDV) is a novel Megalocytivirus causing scale drop disease (SDD) in Asian sea bass in Southeast Asia. In order to support disease diagnosis and surveillance, the present study developed a highly sensitive and specific SYBR Green qPCR assay for rapid detection of SDDV. Specific primers targeting a 135-bp fragment of ATPase coding gene and a 121-bp fragment of major capsid protein (MCP) coding gene of the SDDV genome were newly designed. The optimized qPCR conditions using either ATPase or MCP primers showed no cross amplification with DNA extracted from 12 viruses and bacteria commonly found in aquatic animals. However, the former protocol had a detection limit of 2 viral copies per reaction while the latter had 20 copies. Thus, the SDDV ATPase qPCR method was subsequently validated with field samples (n=86). The results revealed that all clinically sick fish (n=34) from 5 affected farms revealed positive results. Interestingly, 30/52 samples of apparently healthy fish from 8 unaffected farms which previously tested negative for SDDV by semi-nested PCR assay were positive by the newly developed qPCR method. This suggested that qPCR method is highly sensitive and suitable for early screening of SDDV from clinically healthy fish and for disease confirmation of sick fish. Investigation of tissue tropism and viral load of SDDV revealed systemic viral infection in all 8 tested organs with viral loads ranging from 8×10 2 to 6.8×10 4 copies per 200 ng of DNA template. The newly developed qPCR method in this study delivered an accurate and reliable method for rapid detection of SDDV that may facilitate active surveillance in Asian sea bass farming industry.