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

Abstract

Lates calcarifer herpes virus (LCHV) is a new virus of farmed barramundi in Southeast Asia. However, a rapid detection method is yet to be available for LCHV. This study, therefore, aimed to develop a rapid quantitative PCR (qPCR) detection method for LCHV and made it timely available to public for disease diagnostics and surveillance in barramundi farming countries. A newly designed primer set targeting a 93-bp fragment of the LCHV putative major envelope protein encoding gene (MEP) was used for developing and optimizing a SYBR Green based qPCR assay. The established protocol could detect as low as 10 viral copies per µl of DNA template in a reaction containing spiked host DNA. No cross-amplification with genomic DNA extracted from host as well as common aquatic pathogens (12 bacteria and 3 viruses) were observed. Validation test of the method with clinical samples revealed that the virus was detected in multiple organs of the clinically sick fish but not in the healthy fish. We thus recommend that barramundi farming countries should promptly initiate active surveillance for LCHV in order to understand their circulation for preventing possibly negative impact to the industry.HighlightsThis study reported a new SYBR Green qPCR method for detection of LCHVThe qPCR method had detection limit of 10 copies per µl plasmid DNA template when spiked with genomic DNA from the hostThe aforementioned method is highly specific to LCHVValidation with clinical samples revealed that LCHV could be detected from multiple organs with fin and brain the best organs for qPCR detection