Recombinase polymerase amplification assay for rapid detection of lumpy skin disease virus.

Mohamed A Shalaby,Donata Hoffmann,Ayman El-Deeb,Mohamed El-Tholoth,Claus-Peter Czerny,Manfred Weidmann,Frank T Hufert,Ahmed Abd El Wahed

doi:10.1186/s12917-016-0875-5

Abstract

BackgroundLumpy skin disease virus (LSDV) is a Capripoxvirus infecting cattle and Buffalos. Lumpy skin disease (LSD) leads to significant economic losses due to hide damage, reduction of milk production, mastitis, infertility and mortalities (10 %). Early detection of the virus is crucial to start appropriate outbreak control measures. Veterinarians rely on the presence of the characteristic clinical signs of LSD. Laboratory diagnostics including virus isolation, sequencing and real-time polymerase chain reaction (PCR) are performed at well-equipped laboratories. In this study, a portable, simple, and rapid recombinase polymerase amplification (RPA) assay for the detection of LSDV-genome for the use on farms was developed.ResultsThe LSDV RPA assay was performed at 42 °C and detected down to 179 DNA copies/reaction in a maximum of 15 min. Unspecific amplification was observed with neither LSDV-negative samples (n = 12) nor nucleic acid preparations from orf virus, bovine papular stomatitis virus, cowpoxvirus, Peste des petits ruminants and Blue tongue virus (serotypes 1, 6 and 8). The clinical sensitivity of the LSDV RPA assay matched 100 % (n = 22) to real-time PCR results. In addition, the LSDV RPA assay detected sheep and goat poxviruses.ConclusionThe LSDV RPA assay is a rapid and sensitive test that could be implemented in field or at quarantine stations for the identification of LSDV infected case.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0875-5) contains supplementary material, which is available to authorized users.

Highlights

Lumpy skin disease virus (LSDV) is a Capripoxvirus infecting cattle and Buffalos
This study describes the development and evaluation of a real-time recombinase polymerase amplification (RPA) assay for the detection of LSDV genome
DNA molecular standards To produce a molecular LSDV DNA standard, a 910 nt fragment of the G-protein-coupled chemokine receptor (GPCR) gene (6981-7891 of the Genbank accession number: AF325528.1) of the LSDV reference strain (Neethling strain provided by the Pirbright Institute to the Friedrich-Loeffler-Institute, Greifswald-Insel Riems, Germany) was amplified using the in-house designed forward primer (FP): 5′-CATAGTCGATATCCCACATT G-3′, the reverse primer (RP): 5′- GCTAATACTACCA GCACTAC-3′ and the Taq DNA Polymerase (5 PRIME GmbH, Hilden, Germany)

Summary

Introduction

Lumpy skin disease virus (LSDV) is a Capripoxvirus infecting cattle and Buffalos. Lumpy skin disease (LSD) leads to significant economic losses due to hide damage, reduction of milk production, mastitis, infertility and mortalities (10 %). Laboratory diagnostics including virus isolation, sequencing and real-time polymerase chain reaction (PCR) are performed at well-equipped laboratories. Lumpy skin disease (LSD) affects primarily cattle and occasionally buffalo [1, 2]. It causes pyrexia, generalized skin and pox lesions of internal organs, as well as generalized lymphadenopathy [3, 4]. It was reported for the first time in Zambia in 1929 as a hypersensitivity

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