An improved visual closed tube Loop mediated isothermal amplification (LAMP) assay for rapid identification of orf virus in sheep and goats.

The orf virus (ORFV) is an epitheliotropic virus causing a highly contagious skin disease mainly in sheep and goats. Several diagnostics including molecular tools like Loop mediated isothermal amplification (LAMP) assay are available to detect ORFV in affected species. However, the carry-over contamination associated with LAMP as open tube format prevents the assay applicability as point of care test in field diagnostic settings. In this study, the B2L gene based LAMP assay was optimized in a closed tube format using hydroxynaphthol blue (HNB) and calcein as pre-addition dyes and it has shown a clear positive and negative signal at 60 °C using 4 and 5 mM concentrations of MgSO4 respectively for these dyes. Optimitimzed assay that could reveal the result within one hour is highly specific and senstive with a limit of detection at 12.5 femtogram of viral genomic DNA or ~85 virus genome equivalent. This improved method prevented the cross-contamination of future LAMP reactions in the laboratory without compromising diagnostic sensitivity (100%) and specificity (100%) when compared to open tube system. This closed tube LAMP method has potential to act as a simple visual detection assay for the rapid and specific diagnosis of ORFV in sheep and goats.