Comparison of a loop-mediated isothermal amplification for orf virus with quantitative real-time PCR

Abstract

Background: Orf virus (ORFV) causes orf (also known as contagious ecthyma or contagious papular dermatitis), a severe infectious skin disease in goats, sheep and other ruminants. Therefore, a rapid, highly specific and accurate method for the diagnosis of ORFV infections is essential to ensure that the appropriate treatments are administered and to reduce economic losses.

Methods: A loop-mediated isothermal amplification (LAMP) assay based on the identification of the F1L gene was developed for the specific detection of ORFV infections. The sensitivity and specificity of the LAMP assay were evaluated, and the effectiveness of this method was compared with that of real-time PCR.

Results: The sensitivity of this assay was determined to be 10 copies of a standard plasmid. Furthermore, no cross-reactivity was found with either capripox virus or FMDV. The LAMP and real-time PCR assays were both able to detect intracutaneous- and cohabitation-infection samples, with a concordance of 97.83%. LAMP demonstrated a sensitivity of 89.13%.

Conclusion: The LAMP assay is a highly efficient and practical method for detecting ORFV infection. This LAMP method shows great potential for monitoring the prevalence of orf, and it could prove to be a powerful supplemental tool for current diagnostic methods.

Figure 1

Analysis of LAMP products. LAMP amplification products were analyzed using agarose gel electrophoresis (A) and visually inspected with SYBR green I dye under daylight conditions against a black background (B). M: DNA marker; 1: Plasmid containing the F1L gene; 2: Genomic DNA of ORFV; 3: DNA from healthy goats; 4: Water.

Figure 2

Sensitivity of LAMP. Agarose gel electrophoresis (A) and visual inspection using SYBR Green I staining of the LAMP products (B). M: DNA marker; 1–9 are the reaction results from a 10-fold serial dilution of plasmid containing the F1L gene from 10⁸ to 10⁰ copies per reaction; 10: Negative control.

Figure 3

Specificity of LAMP. Agarose gel electrophoresis (A) and visual inspection using SYBR Green I staining (B). M: DNA marker; 1: Plasmid containing the F1L gene; 2: Negative control; 3–12: ORFV/HB/CHA, ORFV/Vaccine/CHA, ORFV/Xinjiang/CHA, ORFV/Chongqing/CHA, ORFV/Shanxi/CHA, ORFV/Guangxi/CHA, ORFV/Gansu/CHA, ORFV/Liaoning/CHA, ORFV/Jilin/CHA and ORFV/Sichuan/CHA; 13 and 14: FMDV/O/CHA and FMDV/Asia I/JS; 15 and 16: Capripox virus/China Vaccine and Capripox virus/Henan/CHA.

Figure 4

Sensitivity and specificity of real-time PCR assay. The standard curve (A) and dissociation curve (B) were generated using known concentration of recombinant plasmid containing the B2L gene from 10⁸ to 10⁰ copies. The dissociation curves of real-time PCR used to detect ORFV and other select viruses (C).