AbstractThe 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 for these dyes, respectively. This optimized assay that could reveal the result within one hour is highly specific and sensitive with a limit of detection of 12.5 femtogram of viral genomic DNA or ~ 85 virus genome equivalent. This improved method can also prevent the cross‑contamination of LAMP reactions in the laboratory without compromising diagnostic sensitivity and specificity 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.
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