Development and Validation of a Droplet Digital PCR Assay for Detection of Feline Herpesvirus Type-1

Feline herpesvirus type-1 (FHV-1), a double-stranded DNA virus, which is a highly infectious upper respiratory tract infection of felids, particularly in kittens. Droplet digital PCR (ddPCR) provides an absolute quantification method with high sensitivity and accuracy. This study aimed to develop a highly sensitive and accurate ddPCR assay for the detection of FHV-1. We designed primers and a probe targeting the FHV-1 glycoprotein D (gD) gene and evaluated the assay's limit of detection (LOD), sensitivity, repeatability, and specificity in comparison to quantitative real-time PCR (qPCR). The developed ddPCR assay demonstrated a strong linear dynamic range (R² ≥ 0.99) and an exceptionally low LOD of 0.18 copies/μL, which was significantly more sensitive than the method qPCR (LOD ~10 copies/μL). Additionally, the assay exhibited high specificity with no cross-reactivity against other common feline pathogens (feline calicivirus, FCV; feline panleukopenia virus, FPV; feline infectious peritonitis virus, FIPV; <i>Bordetella bronchiseptica</i> and <i>Chlamydia felis</i>) and displayed outstanding repeatability (inter-run CV < 1.35). When applied to 118 clinical samples, the ddPCR assay achieved a significantly higher positive detection rate (27.4%) compared to qPCR (14.8%). In conclusion, we have successfully established a reliable ddPCR assay for the absolute quantification of FHV-1, providing a superior tool for laboratory diagnosis and research.