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 × 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

Detection of scale drop disease virus from non‐destructive samples and ectoparasites of Asian sea bass, Lates calcarifer

“…Detection of SDDV in multiple types of samples (seven destructive and three non-destructive) implied that SDDV infection is systemic during a clinical disease outbreak as previously suggested (Sriisan et al, 2020). Moreover, the presence of the virus in blood and mucus of all tested samples suggested that infected fish (both clinically sick and subclinical) from a disease outbreak can shed the virus to both systemic and mucosal systems similar to that of other viruses in finfish such as TiLV in tilapia (Chiamkunakorn et al, 2019;Liamnimitr et al, 2018) and ISAV in salmon (Aamelfot et al, 2015;Giray et al, 2005).…”

“…All fish were alive after sample collection. In order to confirm that the positive sample(s) was truly positive, the positive sample(s) was then assayed with the SYBR Green quantitative polymerase chain reaction (qPCR) method according to Sriisan et al (2020).…”

“…considerable economic loss to farmers (Gibson-Kueh et al, 2012; de Groof et al, 2015;Senapin et al, 2019). Several molecular diagnostic methods for SDDV are currently available including conventional single and semi-nested PCR (Charoenwai et al, 2019;Senapin et al, 2019), quantitative PCR (qPCR) (de Groof et al, 2015;Sriisan et al, 2020) and loop-mediated isothermal amplification (LAMP) (Dangtip et al, 2019). All aforementioned methods relied on destructive sampled fish tissues such as spleen, liver, kidney, brain, eyes and muscle.…”

“…Previous studies reported detection of the virus in multiple organs (e.g. liver, kidney, spleen, gills, brain, eyes and fin), suggesting that SDDV caused systemic infection in Asian sea bass (Senapin et al, 2019;Charoenwai et al, 2019;Sriisan et al, 2020). Previously, Giray et al (2005) reported blood as a reliable non-lethal sample type for the detection of infectious salmon anaemia virus (ISAV) in both moribund and asymptomatic salmon.…”

Impact of transport conditions and underlying disease on post‐stocking survival of juvenile Lates calcarifer

“…Extracted DNA was tested by conventional polymerase chain reaction (cPCR) using published primer sequences and methods for the novel Vibrio spp. that cause "big belly" disease (Gibson-Kueh et al, 2021;Segers & Grisez, 2005), Vibrio harveyi (Pang et al, 2006), Streptococcus iniae (Torres-Corral & Santos, 2021), scale drop disease virus (SDDV) (Sukhontip et al, 2019) Note: Fish were fasted 1-2 days prior to transfers. Neither sedation nor ammonia-binding additives were used during transport.…”