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New real-time PCR assay using allelic discrimination for detection and differentiation of equine herpesvirus-1 strains with A2254 and G2254 polymorphisms.
- Source :
-
Journal of clinical microbiology [J Clin Microbiol] 2012 Jun; Vol. 50 (6), pp. 1981-8. Date of Electronic Publication: 2012 Apr 04. - Publication Year :
- 2012
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Abstract
- A single-nucleotide polymorphism (A(2254) or G(2254)) in open reading frame 30 (ORF30) has been linked to the neuropathogenic phenotype of equine herpesvirus-1 (EHV-1). Identification of this polymorphism led to the development of a real-time PCR (rPCR) assay using allelic discrimination (E(2)) to distinguish between potentially neuropathogenic and nonneuropathogenic EHV-1 strains (G. P. Allen, J. Vet. Diagn. Invest. 19:69-72, 2007). Although this rPCR assay can detect and genotype EHV-1 strains, subsequent studies demonstrated that it lacks the sensitivity for the routine detection of viral nucleic acid in clinical specimens. Therefore, a new allelic discrimination EHV-1 rPCR assay (E(1)) was developed by redesigning primers and probes specific to ORF30. The E(1) and E(2) rPCR assays were evaluated using 76 archived EHV isolates and 433 clinical specimens from cases of suspected EHV-1 infection. Nucleotide sequence analysis of ORF30 was used to confirm the presence of EHV-1 and characterize the genotype (A(2254) or G(2254)) in all archived isolates plus 168 of the clinical samples. The E(1) assay was 10 times more sensitive than E(2), with a lower detection limit of 10 infectious virus particles. Furthermore, all A(2254) and G(2254) genotypes along with samples from three cases of dual infection (A(2254)+G(2254)) were correctly identified by E(1), whereas E(2) produced 20 false dual positive results with only one actual mixed A(2254)+G(2254) genotype confirmed. Based on these findings, E(1) offers greater sensitivity and accuracy for the detection and A/G(2254) genotyping of EHV-1, making this improved rPCR assay a valuable diagnostic tool for investigating outbreaks of EHV-1 infection.
- Subjects :
- Animals
Diagnostic Errors statistics & numerical data
Herpesviridae Infections diagnosis
Herpesviridae Infections virology
Herpesvirus 1, Equid genetics
Herpesvirus 1, Equid pathogenicity
Horse Diseases virology
Horses
Sensitivity and Specificity
Herpesviridae Infections veterinary
Herpesvirus 1, Equid classification
Herpesvirus 1, Equid isolation & purification
Horse Diseases diagnosis
Polymorphism, Single Nucleotide
Real-Time Polymerase Chain Reaction methods
Virology methods
Subjects
Details
- Language :
- English
- ISSN :
- 1098-660X
- Volume :
- 50
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- Journal of clinical microbiology
- Publication Type :
- Academic Journal
- Accession number :
- 22493339
- Full Text :
- https://doi.org/10.1128/JCM.00135-12