Use of an internal standard in a closed one-tube RT-PCR for the detection of equine arteritis virus RNA with fluorescent probes.
Abstract: Routine detection of equine arteritis virus (EAV) can be achieved by virus isolation (VI) in cell culture, or by the amplification of viral genome by molecular methods. To simplify molecular diagnosis, a number of different Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and RT-nested PCR (RT-nPCR) assays were compared, and a one-tube method was developed and optimised utilizing a fluorogenic probe (TaqMan). An artificial RNA template (Mimic) and associated probe were also constructed to provide in-tube validation of the RT-PCR system. To assess the utility of the RT-PCR TaqMan assay, 28 different isolates of EAV representing different genetic groups of American and European strains were tested. Furthermore, the ability of VI and RT-PCR TaqMan assay to detect EAV in different biological matrices such as semen, nasal and faecal swabs and blood was compared. All 28 EAV strains were detected by the RT-PCR TaqMan assay. The results of TaqMan and VI testing were in agreement for 30 of the 33 semen samples and all of the 50 other clinical specimens examined: the RT-PCR TaqMan assay detected 18 positive semen samples, three more than VI. In conclusion, the one-tube RT-PCR TaqMan assay is a rapid, reliable method for the detection of EAV.
Publication Date: 2003-03-27 PubMed ID: 12657208DOI: 10.1051/vetres:2002063Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Diagnosis
- Diagnostic Technique
- Disease control
- Disease Diagnosis
- Disease Management
- Disease Treatment
- Equine Diseases
- Equine Health
- Equine Science
- Equine Viral Arteritis
- Infection
- Infectious Disease
- Laboratory Methods
- Molecular biology
- Polymerase Chain Reaction
- Real-Time PCR
- RNA
- Veterinary Medicine
- Veterinary Research
- Virology
- Virus
Summary
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The research evaluates a more efficient method of detecting equine arteritis virus (EAV) using a one-tube reverse transcriptase-polymerase chain reaction (RT-PCR) with a fluorescent probe. Tests show promising results compared to traditional virus isolation methods.
Comparing Methods of EAV Detection
- This research began with the comparison of different methods for detecting EAV. The standard method is virus isolation in cell culture, but the authors sought to simplify this using molecular techniques and so they examined various kinds of RT-PCR and RT-nested PCR (RT-nPCR) assays.
- With these tests, the authors developed and optimised a one-tube method utilising a specific type of fluorescent probe known as TaqMan.
Creation of an Artificial RNA Template
- A significant part of this work was the construction of an artificial RNA template (or ‘Mimic’) and an associated probe. These elements were used to validate the effectiveness of the RT-PCR system within the same tube, providing a simpler and more streamlined process for detecting EAV.
Testing a Range of EAV Strains
- To determine the functionality and effectiveness of the RT-PCR TaqMan assay, 28 different isolates of EAV were tested. These represented a variety of genetic groups from both American and European strains of EAV.
- The RT-PCR TaqMan assay was able to detect all 28 of the tested strains.
Comparison with Traditional Techniques
- The RT-PCR TaqMan method was also compared to traditional VI techniques. The researchers tested samples from different biological matrices, such as semen, nasal and faecal swabs, and blood. Results both from the traditional VI method and the new TaqMan assay were compared.
- Out of 33 semen samples, TaqMan and VI results agreed in 30 cases. In the 50 other clinical specimens tested, the results agreed completely.
- However, the RT-PCR TaqMan assay was able to identify 3 more positive samples than the VI test, demonstrating a potential for greater accuracy.
Conclusion
- Overall, the results show that the one-tube RT-PCR TaqMan assay is a quick and dependable method for detecting EAV. This method could replace more complex and time-consuming techniques, simplifying the process for detecting this virus.
Cite This Article
APA
Westcott DG, King DP, Drew TW, Nowotny N, Kindermann J, Hannant D, Belák S, Paton DJ.
(2003).
Use of an internal standard in a closed one-tube RT-PCR for the detection of equine arteritis virus RNA with fluorescent probes.
Vet Res, 34(2), 165-176.
https://doi.org/10.1051/vetres:2002063 Publication
Researcher Affiliations
- Department of Virology, Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey, KT15 3NB, UK. d.g.westcott@vla.defra.gsi.gov.uk
MeSH Terms
- Animals
- Arterivirus Infections / diagnosis
- Arterivirus Infections / veterinary
- Arterivirus Infections / virology
- DNA Probes
- Equartevirus / genetics
- Equartevirus / isolation & purification
- Feces / virology
- Fluorescent Dyes
- Horse Diseases / diagnosis
- Horse Diseases / virology
- Horses
- Male
- RNA, Viral / analysis
- RNA, Viral / blood
- RNA, Viral / genetics
- Reference Standards
- Reverse Transcriptase Polymerase Chain Reaction / methods
- Semen / virology
- Sensitivity and Specificity
- Templates, Genetic
Citations
This article has been cited 4 times.- Nagy A, Vitásková E, Černíková L, Křivda V, Jiřincová H, Sedlák K, Horníčková J, Havlíčková M. Evaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay. Sci Rep 2017 Jan 25;7:41392.
- Madi M, Mioulet V, King DP, Lomonossoff GP, Montague NP. Development of a non-infectious encapsidated positive control RNA for molecular assays to detect foot-and-mouth disease virus. J Virol Methods 2015 Aug;220:27-34.
- Balasuriya UB, Go YY, MacLachlan NJ. Equine arteritis virus. Vet Microbiol 2013 Nov 29;167(1-2):93-122.
- Revilla-Fernández S, Wallner B, Truschner K, Benczak A, Brem G, Schmoll F, Mueller M, Steinborn R. The use of endogenous and exogenous reference RNAs for qualitative and quantitative detection of PRRSV in porcine semen. J Virol Methods 2005 Jun;126(1-2):21-30.
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