Abstract
BackgroundSheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are the most serious poxviruses of ruminants. They are double stranded DNA viruses of the genus Capripoxvirus, (subfamily Chordopoxvirinae) within the family Poxviridae. The aim of this study was to develop a Loop-mediated isothermal AMPlification (LAMP) assay for the detection of Capripoxvirus (CaPV) DNA.ResultsA single LAMP assay targeting a conserved region of the CaPV P32 gene was selected from 3 pilot LAMP assays and optimised by adding loop primers to accelerate the reaction time. This LAMP assay successfully detected DNA prepared from representative CaPV isolates (SPPV, GTPV and LSDV), and did not cross-react with DNA extracted from other mammalian poxviruses. The analytical sensitivity of the LAMP assay was determined to be at least 163 DNA copies/μl which is equivalent to the performance reported for diagnostic real-time PCR currently used for the detection of CaPV. LAMP reactions were monitored with an intercalating dye using a real-time PCR machine, or by agarose-gel electrophoresis. Furthermore, dual labelled LAMP products (generated using internal LAMP primers that were conjugated with either biotin or fluorescein) could be readily visualised using a lateral-flow device.ConclusionsThis study provides a simple and rapid approach to detect CaPV DNA that may have utility for use in the field, or in non-specialised laboratories where expensive equipment is not available.
Highlights
Sheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are the most serious poxviruses of ruminants
LSDV occurs across Africa and in recent years the virus has been found in several countries of the Middle East [4]
Optimisation of the Capripoxvirus Loop-mediated isothermal AMPlification (LAMP) assay In initial experiments, all three candidate LAMP assays that targeted the RNA polymerase subunit RPO30, DNA topoisomerase I and the P32 regions generated characteristic laddering patterns after agarose-gel electrophoresis (Figure 1A) and an increase in fluorescence in a real-time polymerase chain reaction (PCR) machine (Figure 1B)
Summary
Sheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are the most serious poxviruses of ruminants. Sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV) cause serious pox diseases of domesticated ruminants [1] They are large, complex, double-stranded DNA viruses of the genus Capripoxvirus, subfamily Chordopoxvirinae, family Poxviridae [2]. A new group of nucleic acid detection assays that exploit isothermal amplification mechanisms have been developed as potential diagnostic tools for use in either the field or low cost laboratory settings These assay formats include loop-mediated isothermal amplification (LAMP) [14] which is a DNA-dependent amplification method that uses a combination of four to six primers targeting six to eight genomic regions, whilst utilising the activity of a strand displacing DNA polymerase. The aim of this study was to develop and evaluate a LAMP assay for the detection of CaPV DNA and to evaluate this assay using different detection formats that might be suitable for use in simple laboratories
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