Development of loop-mediated isothermal amplification assay for specific and rapid detection of camelpox virus in clinical samples

► The LAMP assay targeting ankyrin repeat protein gene (C18L) was developed for simple and rapid detection of camelpox virus. ► The assay was found to be specific only for CMLV genome. ► The detection limit of CMLV-LAMP was thirty copies per reaction and 10 times more sensitive than conventional PCR. ► Comparative sensitivities of the conventional PCR, real time PCR and the LAMP assays were 76.4%, 94.1% and 88.2% respectively. In this study, development of loop-mediated isothermal amplification (LAMP) assay based on ankyrin repeat protein gene (C18L) for specific and rapid detection of camelpox virus (CMLV) was carried out. The assay was optimized using viral genomic DNA (gDNA) extracted from density gradient purified CMLV and standard control recombinant DNA plasmid containing the target, which resulted in reliable amplification at 62°C for 60min. The amplified LAMP product was identified by agarose gel electrophoresis and subsequent direct visualization under UV light or observation by naked-eye for the presence of turbidity and color change following the addition of SYBR Green I dye and hydroxy naphthol blue (HNB). The analytical specificity of LAMP and conventional PCR assays was evaluated using other related poxviruses namely buffalopox, goatpox, sheeppox, and orf viruses, which revealed only a specific amplification of CMLV. The LAMP assay was 10-fold more sensitive than the conventional PCR. Further, the assay was evaluated with DNA extracted from the cell culture isolates of CMLV (n=11) and clinical samples (n=23). These results proved that the developed LAMP is a simple, specific, sensitive, rapid and economical diagnostic tool for detection of CMLV from clinical materials.