Single-Tube Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Red-Spotted Grouper Nervous Necrosis Virus in Fish Species.

Betanodavirus is a causative agent of viral nervous necrosis (VNN) and a major threat to marine and brackish-water aquaculture globally. This virus causes epizootic outbreaks with particularly high morbidity and mortality in larval and juvenile stages and causes significant economic losses in aquaculture. Here, we developed a rapid and highly sensitive single-tube Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) assay for the detection of red-spotted grouper nervous necrosis virus (RGNNV) genotype infection in fish tissue samples. Six primers targeting eight conserved regions of the RNA2 coat protein gene of RGNNV were designed with conservation regions across RGNNV genotypes. RT-LAMP assay was completed within 60 min at 65 °C using a single-tube format that combined reverse transcription and isothermal amplification, and results were directly visualized by the addition of SYBR Green I dye, producing a colour change from orange (negative) to green (positive), observable with the naked eye or under UV illumination. The developed RT-LAMP assay was able to detect five copies of RGNNV from infected samples, which was 20-fold more sensitive than conventional reverse transcription-PCR. The assay demonstrated diagnostic sensitivity and specificity in two fish hosts (Asian seabass and cobia) and showed no cross reactivity with other fish viruses such as tilapia lake virus and cyprinid herpesvirus-2. The developed assay is simple, cost-effective, specific, and enables rapid detection of RGNNV in fish tissues. This single-tube RT-LAMP assay can be applied to screening broodstock facilities, fingerlings, aquaculture farms, quarantine facilities, and juveniles before stocking in ponds or cages, helping prevent disease outbreaks and the transmission of RGNNV in aquaculture systems.