New real-time PCR assay using allelic discrimination for detection and differentiation of equine herpesvirus-1 strains with A2254 and G 2254 polymorphisms

Kathryn L. Smith, Yanqiu Li, Patrick Breheny, R. Frank Cook, Pamela J. Henney, Stephen Sells, Stéphane Pronost, Zhengchun Lu, Beate Crossley, Peter J. Timoney, Udeni B R Balasuriya

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

A single-nucleotide polymorphism (A2254 or G2254) 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 (E1) was developed by redesigning primers and probes specific to ORF30. The E1 and E2 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 G2254) in all archived isolates plus 168 of the clinical samples. The E1 assay was 10 times more sensitive than E2, with a lower detection limit of 10 infectious virus particles. Furthermore, all A2254 and G2254 genotypes along with samples from three cases of dual infection (A2254+G2254) were correctly identified by E1, whereas E2 produced 20 false dual positive results with only one actual mixed A2254+G 2254 genotype confirmed. Based on these findings, E1 offers greater sensitivity and accuracy for the detection and A/G2254 genotyping of EHV-1, making this improved rPCR assay a valuable diagnostic tool for investigating outbreaks of EHV-1 infection.

Original languageEnglish (US)
Pages (from-to)1981-1988
Number of pages8
JournalJournal of Clinical Microbiology
Volume50
Issue number6
DOIs
StatePublished - Jun 2012

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Equid Herpesvirus 1
Real-Time Polymerase Chain Reaction
Genotype
Open Reading Frames
Herpesviridae Infections
Virion
Nucleic Acids
Single Nucleotide Polymorphism
Disease Outbreaks
Sequence Analysis
Limit of Detection
Phenotype

ASJC Scopus subject areas

  • Microbiology (medical)

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New real-time PCR assay using allelic discrimination for detection and differentiation of equine herpesvirus-1 strains with A2254 and G 2254 polymorphisms. / Smith, Kathryn L.; Li, Yanqiu; Breheny, Patrick; Cook, R. Frank; Henney, Pamela J.; Sells, Stephen; Pronost, Stéphane; Lu, Zhengchun; Crossley, Beate; Timoney, Peter J.; Balasuriya, Udeni B R.

In: Journal of Clinical Microbiology, Vol. 50, No. 6, 06.2012, p. 1981-1988.

Research output: Contribution to journalArticle

Smith, Kathryn L. ; Li, Yanqiu ; Breheny, Patrick ; Cook, R. Frank ; Henney, Pamela J. ; Sells, Stephen ; Pronost, Stéphane ; Lu, Zhengchun ; Crossley, Beate ; Timoney, Peter J. ; Balasuriya, Udeni B R. / New real-time PCR assay using allelic discrimination for detection and differentiation of equine herpesvirus-1 strains with A2254 and G 2254 polymorphisms. In: Journal of Clinical Microbiology. 2012 ; Vol. 50, No. 6. pp. 1981-1988.
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abstract = "A single-nucleotide polymorphism (A2254 or G2254) 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 (E1) was developed by redesigning primers and probes specific to ORF30. The E1 and E2 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 G2254) in all archived isolates plus 168 of the clinical samples. The E1 assay was 10 times more sensitive than E2, with a lower detection limit of 10 infectious virus particles. Furthermore, all A2254 and G2254 genotypes along with samples from three cases of dual infection (A2254+G2254) were correctly identified by E1, whereas E2 produced 20 false dual positive results with only one actual mixed A2254+G 2254 genotype confirmed. Based on these findings, E1 offers greater sensitivity and accuracy for the detection and A/G2254 genotyping of EHV-1, making this improved rPCR assay a valuable diagnostic tool for investigating outbreaks of EHV-1 infection.",
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AU - Sells, Stephen

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