Comparison of different molecular methods for assessment of equine arteritis virus (EAV) infection: a novel one-step MGB real-time RT-PCR assay, PCR-ELISA and classical RT-PCR for detection of highly diverse sequences of Slovenian EAV variants.
Abstract: In the present study, a new one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) strategy with minor-groove-binder (MGB) technology for the detection of EAV from 40 semen samples of Slovenian carrier stallions was tested. A novel MGB probe (EAVMGBpr) and a reverse primer (EAV-R) based on the multiple sequence alignment of 49 different EAV strain sequences of the highly conserved ORF7 (nucleocapsid gene) were designed. The performance of the assay was compared with different molecular detection methods. Three different primer pairs targeting the ORF1b and ORF7 were used, respectively. The real-time RT-PCR assay was at least 2 log(10) more sensitive than the classical RT-PCR and at least 1 log(10) more sensitive than the primer set used in the semi-nested PCR. The specificities of the amplification reactions were confirmed with biotinylated probes in the PCR-enzyme-linked immunosorbent assay (PCR-ELISA). Under the conditions described in our study, the sensitivity of the real-time RT-PCR was found to be superior to the PCR-ELISA assay. Thus, while the PCR-ELISA method was found to be both relatively demanding and time consuming, better sensitivity coupled with high specificity and speed of the assay makes the real-time RT-PCR a valuable tool for diagnosis of EAV infection.
Publication Date: 2007-09-12 PubMed ID: 17854913DOI: 10.1016/j.jviromet.2007.07.019Google Scholar: Lookup
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- Comparative Study
- Evaluation Study
- Journal Article
- Research Support
- Non-U.S. Gov't
- Comparative Study
- Diagnosis
- Diagnostic Technique
- Disease
- Disease Diagnosis
- Enzyme-Linked Immunosorbent Assay (ELISA)
- Epidemiology
- Equine Health
- Equine Viral Arteritis
- Genetics
- Horses
- Infection
- Infectious Disease
- Laboratory Methods
- Molecular biology
- Polymerase Chain Reaction
- Real-Time PCR
- Semen Analysis
- Stallion
- Veterinary Medicine
- Virology
Summary
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This research focused on testing and comparing different methods for detecting Equine Arteritis Virus (EAV) infection, with a particular emphasis on a new one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) procedure using MGB technology.
Background and Aim of the Study
- The goal of the study was to test a new RT-PCR methodology for detecting EAV infection from samples taken from carrier stallions.
- Equine Arteritis Virus (EAV) is a virus that can cause disease in horses and significant economic losses in the horse industry.
- The researchers aimed to develop a faster, more sensitive, and specific method of testing for the virus compared to currently available techniques.
Creation of the New Assay
- A novel Minor Groove Binder (MGB) probe and a reverse primer, based on multiple sequence alignment of 49 different EAV strain sequences, were designed.
- The MGB probe and reverse primer targeted the highly conserved ORF7 gene, commonly found across all EAV strains.
Comparison on Sensitivity
- The performance of the new real-time RT-PCR assay was compared to classical RT-PCR and the semi-nested PCR.
- The real-time RT-PCR assay was found to be at least 2 log(10) more sensitive than the classical RT-PCR.
- It was also at least 1 log(10) more sensitive than the primer set used in the semi-nested PCR.
Specificity Analysis
- The specificity of each test was confirmed using biotinylated probes in a PCR-enzyme-linked immunosorbent assay (PCR-ELISA).
- Under conditions described in the study, real-time RT-PCR showed better sensitivity than PCR-ELISA.
Conclusions of the study
- The authors concluded that real-time RT-PCR is an excellent diagnostic tool for EAV due to its sensitivity, specificity, and speed.
- PCR-ELISA, while effective, was deemed relatively demanding and time-consuming compared to the real-time RT-PCR assay.
Cite This Article
APA
Mankoc S, Hostnik P, Grom J, Toplak I, Klobucar I, Kosec M, Barlic-Maganja D.
(2007).
Comparison of different molecular methods for assessment of equine arteritis virus (EAV) infection: a novel one-step MGB real-time RT-PCR assay, PCR-ELISA and classical RT-PCR for detection of highly diverse sequences of Slovenian EAV variants.
J Virol Methods, 146(1-2), 341-354.
https://doi.org/10.1016/j.jviromet.2007.07.019 Publication
Researcher Affiliations
- Virology Unit, Institute for Microbiology and Parasitology, Veterinary Faculty, University of Ljubljana, Gerbiceva 60, SI-1115 Ljubljana, Slovenia. sara.mankoc@vf.uni-lj.si
MeSH Terms
- Animals
- Arterivirus Infections / diagnosis
- Arterivirus Infections / veterinary
- Arterivirus Infections / virology
- Base Sequence
- Carrier State / diagnosis
- Carrier State / veterinary
- Carrier State / virology
- Enzyme-Linked Immunosorbent Assay / methods
- Equartevirus / classification
- Equartevirus / genetics
- Equartevirus / isolation & purification
- Horse Diseases / diagnosis
- Horse Diseases / virology
- Horses
- Male
- Molecular Sequence Data
- Open Reading Frames
- Phylogeny
- Reverse Transcriptase Polymerase Chain Reaction / methods
- Semen / virology
- Sensitivity and Specificity
- Slovenia
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