Development and application of a quadruplex nanoplate-based RT–dPCR assay for the simultaneous detection of canine parvovirus, canine coronavirus, canine distemper virus, and giant panda rotavirus in captive giant pandas

Infection with canine coronavirus (CCoV), canine distemper virus (CDV), canine parvovirus (CPV), and giant panda rotavirus (GPRV) poses important threats to the conservation of giant pandas, especially in juveniles. Given that mixed infections of these viruses are common in giant panda populations, early and accurate differential diagnosis is essential. To detect these four viruses simultaneously, we developed a one-step quadruplex reverse transcription digital PCR (RT–dPCR) assay and compared it with RT–qPCR. We evaluated the specificity, sensitivity, linearity, and repeatability of the assay using recombinant plasmids and nontargeted nucleic acids. Its analytical sensitivity (1.82 copies/μL) was comparable to RT-qPCR on a per-reaction basis (7.28 copies) after adjusting for template volume. The assay was then applied to 475 clinical samples, including 214 giant panda fecal samples, 134 dog rectal swabs samples and 127 cat rectal swabs samples. RT-dPCR outperformed RT-qPCR in detecting positive cases, particularly in canine and feline CPV. Furthermore, all discordant samples positive in RT-dPCR but negative in qPCR were confirmed positive after concentration and RT-qPCR retesting, validating the detection of low viral loads. RT-dPCR also identified four mixed infections that were missed by RT-qPCR, confirming its advantage in multiplex detection. Our results indicate that the RT–dPCR assay exhibited higher analytical sensitivity, detecting more low-load positive samples for the simultaneous detection and quantification of four viral targets in a single reaction and may support early viral surveillance in giant pandas and other susceptible animals.