Development and optimisation of a duplex real-time reverse transcription quantitative PCR assay targeting the VP7 and NS2 genes of African horse sickness virus.
Abstract: Nucleotide sequences of 52 South African isolates of African horse sickness virus (AHSV) collected during 2004-2005 and including viruses of all nine AHSV serotypes, were used to design and develop a duplex real-time reverse transcription quantitative PCR (RT-PCR) assay targeting the VP7 (S8) and NS2 (S9) genes of AHSV. The assay was optimized for detection of AHSV in fresh and frozen blood of naturally infected horses. Assay performance was enhanced using random hexamers rather than gene-specific primers for RT, and with denaturation of double-stranded RNA in the presence of random hexamers. The assay was efficient with a linear range of at least five orders of magnitude. The analytical sensitivity of the assay was 132 copies of the target genes (4125 copies per ml of blood), and the assay was at least 10-fold more sensitive than virus isolation on BHK-21 cells. The assay was also highly specific because it did not detect related orbiviruses, such as bluetongue and equine encephalosis viruses.
Copyright 2010 Elsevier B.V. All rights reserved.
Publication Date: 2010-03-19 PubMed ID: 20304015DOI: 10.1016/j.jviromet.2010.03.009Google Scholar: Lookup
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Summary
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The research developed and improved a diagnostic test that accurately identifies the African horse sickness virus (AHSV) in a horse’s blood samples using real-time reverse transcription quantitative PCR (RT-PCR) technology, targeting two distinctive genes of the AHSV.
Research Context
- African horse sickness (AHS) is a lethal viral disease affecting horses, caused by the African horse sickness virus (AHSV).
- For this research, the researchers used nucleotide sequences of 52 South African strains of AHSV collected during 2004-2005, representing all nine known AHSV serotypes.
- The aim of the research was to develop and optimize a diagnostic assay, more specifically, a duplex real-time reverse transcription quantitative PCR (RT-PCR) assay that targets two specific genes of the AHSV: the VP7 (S8) and NS2 (S9) genes.
Methodology
- The assay was developed for the detection of AHSV in fresh and frozen blood samples collected from naturally infected horses.
- The researchers found that using random hexamers as opposed to gene-specific primers for reverse transcription, and denaturing double-stranded RNA in the presence of random hexamers enhanced the performance of the assay.
Results
- The optimized assay proved to be highly efficient, with a linear range of at least five orders of magnitude.
- The assay’s analytical sensitivity was measured at 132 copies of the targeted virus genes, which is equivalent to 4125 copies of the virus per ml of blood.
- Importantly, the sensitivity of the assay was at least 10 times more than the previous virus isolation system using BHK-21 cells, a traditional cell line used for virus detection.
- The specificity of the test was also validated, as it did not detect related orbiviruses such as bluetongue and equine encephalosis viruses, confirming its selectiveness for detecting AHSV only.
Conclusion
- In summary, the research successfully developed and optimized an advanced RT-PCR assay that accurately identifies the presence of AHSV in horse’s blood, displaying significant improvements in terms of sensitivity and specificity compared to the previous diagnostic methods.
Cite This Article
APA
Quan M, Lourens CW, MacLachlan NJ, Gardner IA, Guthrie AJ.
(2010).
Development and optimisation of a duplex real-time reverse transcription quantitative PCR assay targeting the VP7 and NS2 genes of African horse sickness virus.
J Virol Methods, 167(1), 45-52.
https://doi.org/10.1016/j.jviromet.2010.03.009 Publication
Researcher Affiliations
- Equine Research Centre, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa. melvyn.quan@up.ac.za
MeSH Terms
- African Horse Sickness / diagnosis
- African Horse Sickness / virology
- African Horse Sickness Virus / genetics
- African Horse Sickness Virus / isolation & purification
- Animals
- Antigens, Viral / genetics
- Blood / virology
- DNA Primers / genetics
- Horses
- Molecular Sequence Data
- RNA, Viral / genetics
- Reverse Transcriptase Polymerase Chain Reaction / methods
- Sensitivity and Specificity
- Sequence Analysis, DNA
- Viral Core Proteins / genetics
- Viral Nonstructural Proteins / genetics
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