More sensitive droplet digital PCR test for canine distemper virus

Abstract

Canine distemper virus (CDV) poses a substantial threat to diverse carnivorans, leading to systemic and often fatal diseases. Accurate and prompt diagnosis is paramount for effective management and curbing further transmission. This study evaluates the diagnostic performance of droplet digital PCR (ddPCR) in comparison to conventional reverse-transcription (RT-PCR) and quantitative reverse-transcription real-time PCR (RT-qPCR). Seventy-six clinical samples were collected from dogs with CDV symptoms diagnosed by specialized veterinarians, and sixteen samples from apparently healthy individuals. Conventional PCR, quantitative real-time PCR, and ddPCR were deployed, and their diagnostic capabilities were meticulously assessed. DdPCR exhibited heightened analytical sensitivity, reaching a detection limit of 3 copies/μL, whereas RT-qPCR had a detection limit of 86 copies/μL. The comparative analysis between clinical diagnosis and molecular techniques, including RT-PCR and RT-qPCR, demonstrated low concordance, with Kappa coefficients of 0.268 and 0.324, respectively. In contrast, ddPCR showed a moderate concordance, with a Kappa coefficient of 0.477. The sensitivity was 42.4% for RT-PCR, 57.9% for RT-qPCR, and 72.4% for ddPCR, with 100% specificity for all methods. This study underscores ddPCR's superior sensitivity and agreement with clinical CDV diagnosis, even at low viral concentrations, suggesting it as a promising alternative for CDV diagnosis.