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. 2017 Jul 17;14(1):131.
doi: 10.1186/s12985-017-0792-7.

Development of real-time and lateral flow dipstick recombinase polymerase amplification assays for rapid detection of goatpox virus and sheeppox virus

Yang Yang  1 , Xiaodong Qin  1 , Xiangle Zhang  1 , Zhixun Zhao  1 , Wei Zhang  1 , Xueliang Zhu  1 , Guozheng Cong  1 , Yanmin Li  2 , Zhidong Zhang  3
Affiliations

Development of real-time and lateral flow dipstick recombinase polymerase amplification assays for rapid detection of goatpox virus and sheeppox virus

Yang Yang et al. Virol J. .

Abstract

Background: Goatpox virus (GTPV) and sheeppox virus (SPPV), which belong to the Capripoxvirus (CaPV), are economically important pathogens of small ruminants. Therefore, a sensitive, specific and rapid diagnostic assay for detection of GTPV and SPPV is necessary to accurately and promptly control these diseases.

Methods: Recombinase polymerase amplification (RPA) assays combined with a real-time fluorescent detection (real-time RPA assay) and lateral flow dipstick (RPA LFD assay) were developed targeting the CaPV G-protein-coupled chemokine receptor (GPCR) gene, respectively.

Results: The sensitivity of both CaPV real-time RPA assay and CaPV RPA LFD assay were 3 × 102 copies per reaction within 20 min at 38 °C. Both assays were highly specific for CaPV, with no cross-reactions with peste des petits ruminants virus, foot-and-mouth disease virus and Orf virus. The evaluation of the performance of these two assays with clinical sample (n = 107) showed that the CaPV real-time RPA assay and CaPV RPA LFD assay were able to specially detect SPPV or GTPV present in samples of ovine in liver, lung, kidney, spleen, skin and blood.

Conclusions: This study provided a highly time-efficient and simple alternative for rapid detection of GTPV and SPPV.

Keywords: CaPV RPA LFD; CaPV real-time RPA; Goatpox virus; Recombinase polymerase amplification; Sheeppox virus.

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Conflict of interest statement

Ethics approval and consent to participate

This work was approved by the Animal Ethics Committee of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (approval number LVRIAEC 2012–018). Tissue samples were collected from sheep and goat based on good animal practices of the Animal Ethics Procedures and Guidelines of the People’s Republic of China (AEPGPRC). Tissue samples were collected from the livestock with owner consent as routine disease surveillance. All study participants provided written and informed consent.

Consent for publication

Written informed consents for publication have been obtained from all the participants.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Optimal primers and probe combinations of CaPV real-time RPA assay. Three forward primers (CaPV Fe1 to CaPV Fe3), three reverse primers (CaPV Re1 to CaPV Re3) and one probe (CaPV Pe) were used to select the best combination. NC represents negative control
Fig. 2
Fig. 2
Sensitivity of real-time RPA assay (a) Typical raw fluorescence data of CaPV real-time RPA assay using a dilution series of the pCaPV/RPA DNA. NC represents negative control; (b) Reproducibility of the CaPV real-time RPA assay; (c) The limit of detection in 95% probability based on eight replicates
Fig. 3
Fig. 3
Comparison between performances of CaPV real-time RPA assay with real-time qPCR assay. The correction of CaPV real-time RPA assay threshold time (y axis) with CaPV real-time qPCR assay cycle threshold (CT) values (x axis) on CaPV spiked samples (n = 24) were generated by Excel software
Fig. 4
Fig. 4
Determination of reaction temperature and time (a) CaPV RPA LFD assay are performed at different temperatures as shown. b The test line is visible at 38 °C when the amplification time is longer than 10 min
Fig. 5
Fig. 5
Sensitivity of CaPV RPA LFD assay (a) The sensitivity of CaPV RPA LFD assay was performed using a dilution series of the pCaPV/RPA DNA, and NC represents negative control; (b) The limit of detection in 95% probability based on eight replicates of CaPV RPA LFD assay
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