Simple and Rapid Visual Detection Methods of Orf Virus by B2L Gene based Loop-Mediated Isothermal Amplification Assay

Abstract

Comparison of three different visual detection methods namely the presence of turbidity, color change due to addition of SYBR green I and hydroxynaphthol blue has been studied for a loop-mediated isothermal amplification assay (LAMP) in rapid diagnosis of orf virus as an alternate to gel based analysis. This orf virus specific LAMP assay targeted the amplification of B2L gene sequence of the virus genome and shown specific amplification within 45 min at 60ºC without any cross reactivity to other viruses of sheep and goats. Analytical specificity and sensitivity of the assay were evaluated by these visual detection methods and positive detection rates by LAMP and PCR assays over testing of clinical samples and cell culture adapted virus isolates were determined. B2L LAMP assay detected thirty-five (85.3%) samples, whereas the conventional PCR shown only thirty-three (80.5%) samples as positive from a total of forty one clinical samples tested. On analysis of these thirty five positive samples by three visual detection methods, it is found that HNB and SYBR green I dyes are equally sensitive (100%) and higher compared to turbidity method (94%) of monitoring the LAMP reaction. Use of HNB dye in B2L LAMP assay will be affordable in terms of cost involved and ease of visualization and can suit the less equipped field laboratories for rapid clinical diagnosis of orf virus in sheep and goats.