A sensitive and specific SYBR Green-based qPCR assay for detecting scale drop disease virus (SDDV) in Asian sea bass

Abstract

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.

Development of a SYBR Green quantitative PCR assay for detection ofLates calcarifer herpesvirus(LCHV) in farmed barramundi

“…Detection of LCHV in multiple organs of the clinically sick fish suggests that the virus caused systemic infection, similar to that of SDDV (Senapin et al, 2019; Charoenwai et al, 2019; Sriisan et al, 2020). Interestingly, the liver and kidney tissues which are normally used for PCR diagnostics of fish viruses appeared to be unsuitable for LCHV detection while the fin seemed to be the best targeted tissue due to its highest viral loads, followed by gills, muscle, spleen and brain.…”

“…The optimized qPCR protocol was subsequently used to test for specificity against extracted genomic DNA from i) clinically healthy fish, ii) from 12 common aquatic bacterial species, and iii) from fish samples infected with either infectious spleen and kidney necrosis virus (ISKNV), nervous necrosis virus (NNV), or scale drop disease virus (SDDV). Sample sources and preparation were previously described (Charoenwai et al, 2019; Sriisan et al, 2020). DNA extracted from fin of LCHV-infected fish was used as positive control.…”

“…Therefore, molecular detection methods are required for diagnostic and screening purposes. Several DNA-based detection methods for SDDV have been freely available such as single PCR (Senapin et al, 2019), semi-nested PCR (Charoenwai et al, 2019), loop-mediated isothermal amplification (LAMP) (Dangtip et al, 2019), probe-based qPCR (de Groof et al, 2015), and SYBR Green-based qPCR (Sriisan et al, 2020). The latter one is the most sensitive method with a detection limit of 2 copies of DNA template per reaction.…”

Scale Drop Disease Virus (SDDV) and Lates calcarifer Herpes Virus (LCHV) Coinfection Downregulate Immune-Relevant Pathways and Cause Splenic and Kidney Necrosis in Barramundi Under Commercial Farming Conditions

“…Copy numbers of SDDV and LCHV were calculated from respective standard graphs generated by qPCR amplifications of serially diluted plasmid containing corresponding viral insert target as previously described (Meemetta et al, 2020;Sriisan et al, 2020).…”

“…SDDV and LCHV infections in barramundi reportedly caused similar gross signs of scale loss (Chang et al, 2018), reiterating the need to use more specific diagnostic tests. PCR-based diagnostic methods have been established for these pathogens (Gias et al, 2011;de Groof et al, 2015;Meemetta et al, 2020;Sriisan et al, 2020). However, epidemiological studies characterizing the prevalence of these pathogens in farming sites have not yet been carried out.…”