Development of loop-mediated isothermal amplification and recombinase polymerase amplification combined with lateral flow dipstick assay for the rapid detection of Salmon gill poxvirus.

Abstract

In Atlantic salmon hatcheries, Salmon gill poxvirus (SGPV) can cause parachute, high mortality that can be alleviated by immediate countermeasures. However, this is hampered by a lack of good rapid diagnostics for SGPV. In this study, we developed and evaluated two rapid non-invasive isothermal amplification assays, loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), each coupled with a lateral flow device (LFD) for detection of SGPV in gill mucus or lysates from SGPV-infected salmon. Both assays demonstrated high analytical specificity and strong agreement with the reference real-time PCR assay when purified DNA was tested. The RPA-LFD assay achieved sensitivity comparable to real-time PCR but was consistently inhibited by crude gill mucus, limiting its suitability for direct field testing. In contrast, the LAMP-LFD assay maintained high sensitivity and reproducibility using crude gill mucus without prior nucleic acid extraction, detecting SGPV in 14 of 15 field samples with excellent agreement with real-time PCR. Although analytical sensitivity of LAMP was approximately fourfold lower than real-time PCR, this did not compromise SGPV detection in relevant samples and a final mucus concentration of approximately 4% was optimal for SGPV amplification. Both assays showed no cross-reactivity with common salmon gill pathogens, including Candidatus Branchiomonas cysticola, Desmozoon lepeophtherii, and Paramoeba perurans. The LAMP-LFD workflow provides visual results within 45minutes, representing a viable, rapid, and non-invasive diagnostic platform for field surveillance. These findings demonstrate that LAMP-LFD is well suited for SGPV detection and has strong potential to improve early outbreak detection and enhance the management of SGPV in salmon aquaculture.