Comparative analysis of 3 qPCR primer-probe sets for the detection of equid alphaherpesvirus 1

Yoshinori Kambayashi ¹, Hiroshi Bannai ¹, Manabu Nemoto ¹

⁰Molecular Biology Division, Equine Research Institute, Japan Racing Association, Tochigi, Japan.

Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc +

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October 7, 2025

View abstract on PubMed

Summary

The gB1H qPCR primer-probe set is the most reliable for detecting equid alphaherpesvirus 1 (EqAHV1), offering high sensitivity and specificity. This set surpasses other tested methods for accurate EqAHV1 diagnosis in horses.

Area Of Science

Veterinary Virology
Molecular Diagnostics
Equine Infectious Diseases

Background

Equid alphaherpesvirus 1 (EqAHV1), previously known as equine herpesvirus 1 (EHV1), is a significant pathogen in horses.
The World Organisation for Animal Health (WOAH) Terrestrial Manual has updated its recommendations for EqAHV1 detection.
Three new qPCR primer-probe sets were introduced for EqAHV1 detection, necessitating a comparative evaluation.

Purpose Of The Study

To compare the sensitivity and specificity of three recommended qPCR primer-probe sets for EqAHV1 detection.
To identify the most reliable qPCR assay for accurate diagnosis of EqAHV1 infections.
To evaluate the suitability of primer-probe sets targeting glycoprotein B (gB) and glycoprotein C (gC) genes.

Main Methods

Comparative analysis of three qPCR primer-probe sets (gB1H, gB1P, gC1) against varying EqAHV1 DNA concentrations.
Assessment of specificity by testing cross-reactivity with equid alphaherpesvirus 4 (EqAHV4).
Validation using 120 nasal swab samples from horses with clinical signs of EqAHV1 infection.

Main Results

Sets gB1H and gB1P demonstrated high sensitivity, detecting as few as 10 EqAHV1 DNA copies.
Set gC1 exhibited lower sensitivity, failing to detect ≤10 copies, even after concentration adjustments.
Set gB1P showed cross-reactivity with EqAHV4, compromising its specificity for EqAHV1 detection.
Set gB1H exhibited slightly higher sensitivity than the previously recommended gB1D set.
Set gB1H proved to be the most reliable, offering high sensitivity and specificity for EqAHV1 detection.

Conclusions

The gB1H qPCR primer-probe set is recommended as the most reliable method for EqAHV1 detection.
The gC1 set is less sensitive and not recommended for routine EqAHV1 diagnosis.
Laboratories should validate the gB1H method under their specific conditions prior to implementation.

Keywords:

EHV1 EqAHV1 WOAH Terrestrial Manual equine rhinopneumonitis qPCR