Development of a nanoparticle-assisted PCR assay to distinguish canine coronaviruses I and II

Nanoparticle-assisted PCR (nanoPCR) is a novel method for the simple, rapid, and specific detection of viruses. We developed a nanoPCR method to detect and differentiate canine coronavirus I (CCoV I) and II (CCoV II). Primer pairs were designed against the M gene conserved region of CCoV I and CCoV II, producing specific fragments of 239 bp (CCoV I) and 105 bp (CCoV II). We optimized the annealing temperature and primer concentrations for the CCoV nanoPCR assay and assessed its sensitivity and specificity. Under optimized nanoPCR reaction conditions, the detection limits were 6.47 × 10¹ copies/μL for CCoV I and 6.91 × 10² copies/μL for CCoV II. No fragments were amplified using other canine viruses as templates. The sensitivity of the nanoPCR assay was 100-fold higher than that of a conventional RT-PCR assay. Among 60 clinical samples collected from Beijing, China, the assay detected 12% positive for CCoV I and 48% positive for CCoV II. Our nanoPCR method is an effective method to rapidly detect CCoV I and CCoV II alone, or as a mixed infection, in dogs.