Development of the isothermal recombinase polymerase amplification assays for rapid detection of the genus Capripoxvirus

Libing Liu; Jinfeng Wang; Fuping Nie; Ruiwen Li; Yixiang Gao; Xiaoxia Sun; Wanzhe Yuan; Jianchang Wang

doi:10.1016/j.jviromet.2023.114788

Development of the isothermal recombinase polymerase amplification assays for rapid detection of the genus Capripoxvirus

J Virol Methods. 2023 Oct:320:114788. doi: 10.1016/j.jviromet.2023.114788. Epub 2023 Jul 28.

Authors

Libing Liu¹, Jinfeng Wang¹, Fuping Nie², Ruiwen Li³, Yixiang Gao¹, Xiaoxia Sun¹, Wanzhe Yuan³, Jianchang Wang⁴

Affiliations

¹ Technology Center of Shijiazhuang Customs, Shijiazhuang 050051, China.
² Technology Center of Chongqing Customs, Chongqing 400020, China.
³ College of Veterinary Medicine, Hebei Agricultural University, Baoding 071000, China.
⁴ Technology Center of Shijiazhuang Customs, Shijiazhuang 050051, China. Electronic address: jianchangwang1225@126.com.

PMID: 37517457
DOI: 10.1016/j.jviromet.2023.114788

Abstract

Sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV) belong to the genus Capripoxvirus (CaPV), and are important pathogens of sheep, goat and cattle, respectively. Rapid and reliable detection of CaPV is critical to prevent its spread and promote its eradication. This study aimed to develop the recombinase polymerase amplification (RPA) assays combined with real-time fluorescence (real-time RPA) and naked-eye visible lateral flow strip (LFS RPA) for rapid detection of CaPV. Both developed RPA assays worked well at 39 °C within 20 min. They were highly specific for the detection of GTPV, SPPV and LSDV, while no cross-reactivity was observed for other non-targeted pathogens and genomic DNA of goat, sheep and cattle. The limit of detection for real-time RPA and LFS RPA were 1.0 × 10² and 1.0 × 10¹ copies per reaction, respectively. In the artificially contaminated samples with GTPV, the detection results of RPA assays were consistent with those of real-time PCR. For 15 clinical samples, LSDV was detected by real-time RPA, LFS RPA and real-time PCR in 13, 15 and 15 samples, respectively. The developed RPA assays were specific, sensitive, and user-friendly for the rapid detection of CaPV, and could be a better alternative method applied in low-resources settings.

Keywords: Capripoxvirus; Isothermal amplification; LFS RPA; P32 gene; Real-time RPA.

Publication types

Evaluation Study
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Capripoxvirus* / genetics
Capripoxvirus* / isolation & purification
Cattle
Goats
Nucleic Acid Amplification Techniques* / methods
Poxviridae Infections* / veterinary
Poxviridae Infections* / virology
Recombinases
Sensitivity and Specificity
Sheep
Viral Proteins / genetics

Substances

Recombinases
Viral Proteins