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Comparative Study
. 2015 Dec 2:12:206.
doi: 10.1186/s12985-015-0440-z.

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

Yang Yang  1 , Xiaodong Qin  2 , Guangxiang Wang  3 , Yuen Zhang  4   5 , Youjun Shang  6 , Zhidong Zhang  7
Affiliations
Comparative Study

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

Yang Yang et al. Virol J. .

Abstract

Background: Orf 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.

Results: In 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.

Conclusions: These results suggest that the developed ORFV exo RPA assay is suitable for rapid detection of ORFV.

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Figures

Fig. 1
Fig. 1
Performance of the ORFV exo RPA assay. a Amplification curve of ORFV exo RPA assay over time using a dilution range of 106 to 101 copies/reaction of ORFV. NC represent negative control. b Reproducibility of the ORFV exo RPA assay. The threshold time is represented as the mean ± standard deviation (SD). The standard regression line was generated based on 8 data sets (c) Probit regression analysis using Statistics software was done on data from the eight runs of ORFV exo RPA assay. The limit of detection at 95 % probability is depicted by a triangle
Fig. 2
Fig. 2
Comparison between performances of ORFV exo RPA assay and real-time ORFV qPCR assay on samples of ORFV-infected cells (n = 15) and spiked tissues lysates (n = 24). Linear regression analysis of the exo RPA threshold time (y axis) and qPCR cycle threshold (CT) values (x axis) were determined by Excel software
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