Development of a fluorescent probe-based recombinase polymerase amplification assay for rapid detection of Orf virus.

Yang Yang,Yuen Zhang,Youjun Shang,Zhidong Zhang,Guangxiang Wang,Xiaodong Qin

doi:10.1186/s12985-015-0440-z

Yang Yang, Yuen Zhang + Show 4 more

Open Access

https://doi.org/10.1186/s12985-015-0440-z

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Abstract

BackgroundOrf virus (ORFV) is the causative agent of Orf (also known as contagious ecthyma or contagious papular dermatitis), a severe infectious skin disease in goats, sheep and other ruminants. The rapid detection of ORFV is of great importance in disease control and highly needed. A isothermal molecular diagnostic approach, termed recombinase polymerase amplification (RPA), is considered as an novel and rapid alternative techonology to PCR assay.ResultsIn the present study, a novel fluorescent probe based on RPA assay (ORFV exo RPA assay) was developed. The developed ORFV exo RPA assay was capable of as low as 100 copies of ORFV DNA /reaction and was highly specific, with no cross-reaction with closely related viruses (capripox virus, foot-and-mouth disease virus or peste des petits ruminants virus). Further assessment with clinical samples showed that the developed ORFV exo RPA assay has good correlation with qPCR assays for detection of ORFV.ConclusionsThese results suggest that the developed ORFV exo RPA assay is suitable for rapid detection of ORFV.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0440-z) contains supplementary material, which is available to authorized users.

Highlights

Orf virus (ORFV) is the causative agent of Orf, a severe infectious skin disease in goats, sheep and other ruminants
Samples of other tissues were chosen because various types of tissues are often received in the field diagnostics for differential diagnosis including nonepitheliotropic viruses such as peste des petits ruminants virus (PPRV), so it would be critical to access the compatibility of the developed recombinase polymerase amplification (RPA) assay with different tissues matrix. 10 % (w/vol) tissue suspensions were prepared by homogenizing tissue samples in phosphatebuffered saline (PBS)
We demonstrated that the developed ORFV exo RPA assay could detect ORFV at a copy as low as 100 genome copies/reaction, the results have demonstrated that the developed ORFV RPA exo assay only detects ORFV

Summary

Introduction

Orf virus (ORFV) is the causative agent of Orf ( known as contagious ecthyma or contagious papular dermatitis), a severe infectious skin disease in goats, sheep and other ruminants. The rapid detection of ORFV is of great importance in disease control and highly needed. ORFV is the causative agent of contagious ecthyma or contagious papular dermatitis (Orf ) in goats, sheep and other ruminants, with a worldwide distribution and significant financial importance [1, 2]. Orf is usually more severe in goats than in sheep and the morbidity of the disease may reach up to 100 %. The mortality is usually not high but can reach up to 90 % in susceptible flocks of young sheep in an epidemic situation [5, 6]. The disease has zoonotic potential which adds to the significance of the disease

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