Orf, also known as contagious ecthyma, is a skin disease caused by Orf virus (ORFV), which is classified in the genus Parapoxvirus of the family Poxviridae. Orf principally affects goats and sheep with worldwide distribution and significant financial importance. The clinical symptoms of Orf are characterized by the formation of papules, vesicles and growing scabs on the lips and muzzle of infected animals. ORFV is also a zoonotic parapoxvirus endemic to most countries in the world and is principally associated with small ruminants [
1]. Humans of infection with ORFV is thought to be under diagnosed and causes a public health concern in the context of changing environment and increase in human populations because it can resemble skin lesions associated with potentially life-threatening zoonotic infections such as tularemia, cutaneous anthrax, and erysipeloid. Therefore, rapid and definitive diagnosis is critical and highly needed for confirmation and epidemiological investigations [
1].
At the present, molecular amplification assays, in particular real-time quantitative PCR (qPCR) and loop-mediated isothermal amplification (LAMP) are sensitive for detection of ORFV [
2‐
5]. However, due to the high complexity of assays or dependency of the specialized and rather expensive equipments, none of these tests can be easily adapted to be a Point of care test or are widely used in resource-limited settings where the disease is mostly endemic [
4,
6,
7]. In recent years, a novel and rapid isothermal molecular diagnostic approach, termed recombinase polymerase amplification(RPA), has been developed as an alternative to PCR assay and LAMP [
8]. In this assay, just two target specific oligonucleotide primers are used to bind the template DNA with the assistance of a recombinase in combination with strand-displacement DNA synthesis. Importantly, an amplification of DNA targets can be achieved in less than 30 min at temperatures just above room temperature. Since its initial development in 2006, a different detection format of RPA assays including probe based and lateral flow dipstick (LFD) detection has been successfully developed for rapid detection of various pathogens [
8‐
22]. To meet our need for rapid assay on side and resource-limited settings, a RPA assay in combination with a simpler LFD is a desirable option. Most recently, we successfully developed fluorescent probe-based RPA assay for detection of ORFV with high sensitivity and specificity [
23]. In the present study, a PRA assay in combination with a simpler LFD (designated as ORFV RPA-LFD assay) has been developed and evaluated for rapid detection of ORFV. To the best of our knowledge, it is for the first time a rapid molecular amplification assay was developed for on site detection of ORFV. After determination of the sensitivity and specificity of the assay, clinical samples from sheep were tested and compared with results from the corresponding qPCR assay.