A DPO-Enhanced Gold Nanoparticle-Assisted PCR Assay for Simultaneous Detection of Bovine Viral Diarrhea Virus, Astrovirus, and Rotavirus

To develop a rapid detection tool for major bovine diarrheal viruses, Bovine viral diarrhea virus (BVDV), Bovine astrovirus (BAstV), and Bovine rotavirus (BRV), a gold nanoparticle-enhanced dual-priming oligonucleotide (DPO) multiplex PCR method was established. After optimization, the assay showed high sensitivity with detection limits of 4.9 × 10−1, 2.72 × 102, and 1.88 × 103 copies/μL for BVDV, BAstV, and BRV, respectively, and demonstrated excellent specificity. In the validation using 963 clinical samples, the DPO-nanoPCR showed numerically higher detection rates for BVDV (5.50% vs. 4.36%) and BAstV (1.04% vs. 0.62%) compared to conventional PCR, while the detection rate for BRV was identical (0.93%). Statistical analysis indicated no significant difference between the two methods (p > 0.05), with an overall agreement rate exceeding 98.86%. The developed DPO-nanoPCR method provides a sensitive, specific, and efficient tool for the simultaneous surveillance and diagnosis of these key bovine enteric pathogens.