Scale drop disease virus (SDDV) is a megalocytivirus known to cause disease in Asian sea bass in Southeast Asia. To support SDDV diagnosis and surveillance, we report on a sensitive and specific SYBR Green qPCR assay. The qPCR primers were designed to target a 135 bp fragment of the SDDV ATPase gene. The optimized SDDV qPCR assay reliably detected 2 copies of a plasmid dsDNA control and did not cross-amplify DNA to any of 12 viral or bacterial pathogens commonly found in aquatic animals. When assessed with 86 field samples, the assay detected SDDV in DNA extracted from each of 34 scale drop disease-affected fish collected from 5 affected farms. The qPCR also detected SDDV in DNA from 30 of 52 overtly healthy fish collected from 9 farms where SDDV had not been detected previously, using a semi-nested conventional PCR. The higher sensitivity of our SDDV qPCR assay can thus be useful in detecting fish with subclinical/chronic infections. However, the qPCR showed that SDDV DNA loads varied from 8.0 × 102 to 6.8 × 104 viral DNA copies per 200 ng DNA template among the 8 organ tissue types sampled from 3 diseased fish. In circumstances requiring SDDV to be detected unequivocally in subclinical carriers with lower-level infection, qPCR testing of more than one type of tissue is advisable.

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 × 102 to 6.8 × 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.HighlightsThis study developed a SYBR Green qPCR assay for rapid detection of SDDVThe developed qPCR assay is specific for SDDV with limit of detection of 2 viral copies per reactionThe assay could detect the virus from subclinically infected fish with low viral loadWe recommend this qPCR assay for active surveillance and early screening of SDDV