A highly sensitive and specific SYBR Green quantitative polymerase chain reaction (qPCR) method for rapid detection of scale drop disease virus in Asian sea bass, Lates calcarifer

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 of the SDDV genome were newly designed and subsequent gradient PCR assays were conducted to investigate their optimal annealing temperature. The optimized qPCR assay could detect as low as 2 viral copies per reaction and showed no cross amplification with DNA extracted from 12 viruses and bacteria commonly found in aquatic animals. 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 gave 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 with relatively high viral load (8 x 102 to 6.8 x 104 copies per 200 ng of DNA template) in all 9 tested organs including eyes, brain, fin, gills, kidney, liver, kidney, spleen, and muscle. The newly developed qPCR method in this study delivered an accurate and reliable method for rapid detection of SDDV that may facilitate active surveillance and prevent widespread of the virus. HighlightsO_LIThis study developed a SYBR Green qPCR assay for rapid detection of SDDV C_LIO_LIThe developed qPCR assay is specific for SDDV with limit of detection of 2 viral copies per reaction C_LIO_LIThe assay could detect the virus from subclinically infected fish with low viral load C_LIO_LIWe recommend this qPCR assay for active surveillance and early screening of SDDV C_LI