Abstract
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 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.
References
Adnan F.N. 2011. A rapid, non enzymatic method for genomic DNA extraction from whole blood and mammalian tissues. Res Opin Anim Vet Sci,1(5), 279-283.
Bora D.P., Venkatesan G., Bhanuprakash V., Balamurugan V., Prabhu M., Sankar M.S & Yogisharadhya R. 2011. TaqMan real-time PCR assay based on DNA polymerase gene for rapid detection of Orf infection. J Virol Methods, 178, 249-252.
Gallina L., Dal Pozzo F., Mc Innes C.J., Cardeti G., Guercio A., Battilani M., Ciulli S. & Scagliarini A. 2006. A real time PCR assay for the detection and quantification of orf virus. J Virol Methods, 134, 140-145.
Goto M., Honda E., Ogura A., Nomoto A. & Hanaki K. 2009. Colorimetric detection of the loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechnique, 46, 167–172.
Karki M., Venkatesan G., Kumar A., Kumar S. & Bora D.P. 2019. Contagious ecthyma of sheep and goats: a comprehensive review on epidemiology, immunity, diagnostics and control measures. Vet Arhiv, 89, 393–423.
Karthik K., Rathore R., Thomas P., Arun T.R., Viswas K.N., Dhama K. & Agarwal R.K. 2014. New closed tube loop-mediated isothermal amplification assay for prevention of product cross-contamination. Methods X, 1, 137–143.
Li J., Song D., He W., Bao Y., Lu R., Su G., Wang G., Lu H., Zhao K. & Gao F. 2013. Rapid detection of orf virus by loop-mediated isothermal amplification based on the DNA polymerase gene. Arch Virol, 158,793-798.
Mansour S.M., Ali H., Chase C.C. & Cepica A. 2015. Loop-mediated isothermal amplification for diagnosis of 18 World Organization for Animal Health (OIE) notifiable viral diseases of ruminants, swine and poultry. Anim Health Res Rev, 16, 89-106.
Nandi S., U.K. De & Chowdhury S. 2011. Current status of contagious ecthyma or orf disease in goat and sheep - A global perspective. Small Rumin Res, 96, 73-82.
Notomi T., Okayama H., Masubuchi H., Yonekawa T., Watanabe K., Amino N. & Hase T. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res, 28, e63.
Parida M., Sannarangaiah S., Dash P.K., Rao P.V.L. & Morita K. 2008. Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Rev Med Virol, 18, 407-421.
Quoc N.B., Phuong N.D.N., Chau N.N.B. & Linh D.T.P. 2018. Closed tube loop-mediated isothermal amplification assay for rapid detection of hepatitis B virus in human blood. Heliyon, 64, e00561.
Shome R., Kumar A., Shome B.R., Dubal Z.B., Mukharjee S. & Rahman H. 2005. Outbreak of contagious ecthyma in black Bengal goats in North-East India. Ind Vet J, 82, 241-242.
Tsai S.M., Chan K.W., Hsu W.L., Chang T.J., Wong M.L. & Wang C.Y. 2009. Development of a loop-mediated isothermal amplification for rapid detection of orf virus. J Virol Methods, 157, 200-204.
Venkatesan G., Balamurugan V., Bora D.P., Yogisharadhya R., Prabhu M. & Bhanuprakash V. 2011. Sequence and phylogenetic analyses of an Indian isolate of orf virus from sheep. Vet Ital, 47, 323-332.
Venkatesan G., Bhanuprakash V., Balamurugan V., Kumar A., Bora D.P., Yogisharadhya R., Arya S., Madhvan A., Muthuchelvan D. & Pandey A.B. 2016. Simple and rapid visual detection methods of orf virus by B2L gene based Loop-mediated Isothermal amplification assay. Adv Anim Vet Sci, 4(3), 152-59.
Venkatesan G., Bhanuprakash V., Balamurugan V., Singh R.K. Pandey A.B. 2012. Development of loop-mediated isothermal amplification assay for specific and rapid detection of camelpox virus in clinical samples. J Virol Methods, 183, 34-39.
Venkatesan G., De A., Arya S., Kumar A., Muthuchelvan D., Debnath B.C., Dutta T.K., Hemadri D. & Pandey A.B. 2018. Molecular evidence and phylogenetic analysis of orf virus isolates from outbreaks in Tripura state of North-East India. Virus Dis, 29, 216-220.
Wang K., Shao H., Pei Z. & Hu G. 2016. Rapid detection of contagious ecthyma by loop-mediated isothermal amplification and epidemiology in Jilin Province China. J Vet Med Sci, 78(1),125–128.