Development of a multiplex real-time reverse transcriptase-polymerase chain reaction for equine infectious anemia virus (EIAV).
Abstract: A single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) using a fluorogenic real-time PCR detection method is described for the quantitation of equine infectious anemia virus (EIAV) RNA in the plasma of equids. To compensate for variations inherent in sample preparation a multiplex real-time RT-PCR system was developed that permitted the simultaneous calculation of the nucleic acid recovery rate along with the copy number of viral RNA molecules. Detection of EIAV RNA was linear from 10(9) to 10(1) molecules with intra- and inter-assay variability of less than 1% at 10(8), 10(6), 10(4) and 10(2) molecules.
Publication Date: 2002-08-15 PubMed ID: 12176154DOI: 10.1016/s0166-0934(02)00101-5Google Scholar: Lookup
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- Evaluation Study
- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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The research paper describes the development of a single-use reverse transcriptase-polymerase chain reaction (RT-PCR) for measuring equine infectious anemia virus (EIAV) RNA in horse plasma, with a system incorporated to adjust for variations in sample preparation.
Objective
The objective of this research was to design a reliable and efficient method to quantify equine infectious anemia virus (EIAV) RNA in plasma samples taken from horses. The researchers aimed at accounting for variations that can occur during sample preparation, which can potentially cause variations in the results, by including a system within their method.
Method
- A reverse transcriptase-polymerase chain reaction (RT-PCR) was used for the main part of the research. RT-PCR is a technique that allows for the conversion of RNA into DNA and the amplification of specific DNA targets.
- To allow for a real-time assessment, a fluorescence detection method was incorporated into the RT-PCR. This method provides researchers with the ability to see the results of their assay in real-time, as the amplification process is happening.
- To compensate for potential variations introduced during sample preparation, a multiplex real-time RT-PCR system was developed. This system allows for the simultaneous calculation of the nucleic acid recovery rate – the measure of how much nucleic acid is recovered during the sample preparation process – and the number of viral RNA molecules, thereby correcting any potential inaccuracies in the quantification resulting from preparation variations.
Results
- The system proved to be precise, with detection of EIAV RNA being linear from 10^9 to 10^1 molecules.
- Furthermore, the variability observed, both within and between the assays, was less than 1% at all measured quantities (10^8, 10^6, 10^4, and 10^2 molecules), indicating a consistent and reliable system.
Conclusion
In conclusion, the developed multiplex real-time RT-PCR system was able to accurately quantify equine infectious anemia virus (EIAV) RNA in plasma samples taken from horses. Additionally, it accounted for variations inherent in sample preparation, therefore making it both a reliable and efficient method for the detection and monitoring of EIAV infections.
Cite This Article
APA
Cook RF, Cook SJ, Li FL, Montelaro RC, Issel CJ.
(2002).
Development of a multiplex real-time reverse transcriptase-polymerase chain reaction for equine infectious anemia virus (EIAV).
J Virol Methods, 105(1), 171-179.
https://doi.org/10.1016/s0166-0934(02)00101-5 Publication
Researcher Affiliations
- Department of Veterinary Science, Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546-0099, USA. rfcook@uky.edu
MeSH Terms
- Animals
- DNA Probes
- Equine Infectious Anemia / virology
- Fluorescent Dyes
- Gene Dosage
- Horses
- Infectious Anemia Virus, Equine / genetics
- Infectious Anemia Virus, Equine / isolation & purification
- Infectious Anemia Virus, Equine / physiology
- RNA, Viral / blood
- Reproducibility of Results
- Reverse Transcriptase Polymerase Chain Reaction
- Viral Load
Grant Funding
- R01A12850 / PHS HHS
- R01CA49296 / NCI NIH HHS
Citations
This article has been cited 24 times.- Du C, Duan Y, Wang XF, Lin Y, Na L, Wang X, Chen K, Wang X. Attenuation of Equine Lentivirus Alters Mitochondrial Protein Expression Profile from Inflammation to Apoptosis. J Virol 2019 Nov 1;93(21).
- Brister H, Barnum SM, Reedy S, Chambers TM, Pusterla N. Validation of two multiplex real-time PCR assays based on single nucleotide polymorphisms of the HA1 gene of equine influenza A virus in order to differentiate between clade 1 and clade 2 Florida sublineage isolates. J Vet Diagn Invest 2019 Jan;31(1):137-141.
- Sharav T, Konnai S, Ochirkhuu N, Ts EO, Mekata H, Sakoda Y, Umemura T, Murata S, Chultemdorj T, Ohashi K. Detection and molecular characterization of equine infectious anemia virus in Mongolian horses. J Vet Med Sci 2017 Nov 17;79(11):1884-1888.
- Te SH, Chen EY, Gin KY. Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis. Appl Environ Microbiol 2015 Aug;81(15):5203-11.
- Craigo JK, Ezzelarab C, Cook SJ, Liu C, Horohov D, Issel CJ, Montelaro RC. Protective efficacy of centralized and polyvalent envelope immunogens in an attenuated equine lentivirus vaccine. PLoS Pathog 2015 Jan;11(1):e1004610.
- Liu C, Cook SJ, Craigo JK, Cook FR, Issel CJ, Montelaro RC, Horohov DW. Epitope shifting of gp90-specific cellular immune responses in EIAV-infected ponies. Vet Immunol Immunopathol 2014 Oct 15;161(3-4):161-9.
- Ma J, Wang SS, Lin YZ, Liu HF, Liu Q, Wei HM, Wang XF, Wang YH, Du C, Kong XG, Zhou JH, Wang X. Infection of equine monocyte-derived macrophages with an attenuated equine infectious anemia virus (EIAV) strain induces a strong resistance to the infection by a virulent EIAV strain. Vet Res 2014 Aug 9;45(1):82.
- Singha H, Goyal SK, Malik P, Khurana SK, Singh RK. Development, evaluation, and laboratory validation of immunoassays for the diagnosis of equine infectious anemia (EIA) using recombinant protein produced from a synthetic p26 gene of EIA virus. Indian J Virol 2013 Dec;24(3):349-56.
- Craigo JK, Ezzelarab C, Cook SJ, Chong L, Horohov D, Issel CJ, Montelaro RC. Envelope determinants of equine lentiviral vaccine protection. PLoS One 2013;8(6):e66093.
- Issel CJ, Scicluna MT, Cook SJ, Cook RF, Caprioli A, Ricci I, Rosone F, Craigo JK, Montelaro RC, Autorino GL. Challenges and proposed solutions for more accurate serological diagnosis of equine infectious anaemia. Vet Rec 2013 Feb 23;172(8):210.
- Cappelli K, Capomaccio S, Cook FR, Felicetti M, Marenzoni ML, Coppola G, Verini-Supplizi A, Coletti M, Passamonti F. Molecular detection, epidemiology, and genetic characterization of novel European field isolates of equine infectious anemia virus. J Clin Microbiol 2011 Jan;49(1):27-33.
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- Zhang B, Montelaro RC. Replication of equine infectious anemia virus in engineered mouse NIH 3T3 cells. J Virol 2009 Feb;83(4):2034-7.
- Pawar SN, Mattila JT, Sturgeon TJ, Lin PL, Narayan O, Montelaro RC, Flynn JL. Comparison of the effects of pathogenic simian human immunodeficiency virus strains SHIV-89.6P and SHIV-KU2 in cynomolgus macaques. AIDS Res Hum Retroviruses 2008 Apr;24(4):643-54.
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- Craigo JK, Durkin S, Sturgeon TJ, Tagmyer T, Cook SJ, Issel CJ, Montelaro RC. Immune suppression of challenged vaccinates as a rigorous assessment of sterile protection by lentiviral vaccines. Vaccine 2007 Jan 15;25(5):834-45.
- Stewart M, Desport M, Hartaningsih N, Wilcox G. TaqMan real-time reverse transcription-PCR and JDVp26 antigen capture enzyme-linked immunosorbent assay to quantify Jembrana disease virus load during the acute phase of in vivo infection. J Clin Microbiol 2005 Nov;43(11):5574-80.
- Chung C, Mealey RH, McGuire TC. Evaluation of high functional avidity CTL to Gag epitope clusters in EIAV carrier horses. Virology 2005 Nov 25;342(2):228-39.
- Jin S, Chen C, Montelaro RC. Equine infectious anemia virus Gag p9 function in early steps of virus infection and provirus production. J Virol 2005 Jul;79(14):8793-801.
- Mealey RH, Sharif A, Ellis SA, Littke MH, Leib SR, McGuire TC. Early detection of dominant Env-specific and subdominant Gag-specific CD8+ lymphocytes in equine infectious anemia virus-infected horses using major histocompatibility complex class I/peptide tetrameric complexes. Virology 2005 Aug 15;339(1):110-26.
- Burnouf T, Griffiths E, Padilla A, Seddik S, Stephano MA, Gutiérrez JM. Assessment of the viral safety of antivenoms fractionated from equine plasma. Biologicals 2004 Sep;32(3):115-28.
- Yoshida M, Sai S, Marumo K, Tanaka T, Itano N, Kimata K, Fujii K. Expression analysis of three isoforms of hyaluronan synthase and hyaluronidase in the synovium of knees in osteoarthritis and rheumatoid arthritis by quantitative real-time reverse transcriptase polymerase chain reaction. Arthritis Res Ther 2004;6(6):R514-20.
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