Establishment and application of multiplex droplet digital polymerase chain reaction assay for bovine enterovirus, bovine coronavirus, and bovine rotavirus

Junzhen Chen; Dan Li; Yafang Xu; Zeyu Li; Siqi Ma; Xinyi Liu; Yuanyuan Yuan; Chengyuan Zhang; Qiang Fu; Huijun Shi

doi:10.3389/fvets.2023.1157900

Establishment and application of multiplex droplet digital polymerase chain reaction assay for bovine enterovirus, bovine coronavirus, and bovine rotavirus

Front Vet Sci. 2023 Sep 12:10:1157900. doi: 10.3389/fvets.2023.1157900. eCollection 2023.

Authors

Junzhen Chen¹, Dan Li^{1

2}, Yafang Xu¹, Zeyu Li¹, Siqi Ma¹, Xinyi Liu¹, Yuanyuan Yuan¹, Chengyuan Zhang¹, Qiang Fu¹, Huijun Shi¹

Affiliations

¹ College of Veterinary Medicine, Xinjiang Agricultural University, Ürümqi, China.
² Tecon Biology Co., Ltd., Ürümqi, China.

Abstract

Bovine enterovirus (BEV), bovine coronavirus (BCoV), and bovine rotavirus (BRV) are still the major worldwide concerns in the health care of cattle, causing serious economic losses in the livestock industry. It is urgent to establish specific and sensitive methods to detect viruses for the early control of diseases. Droplet digital PCR (ddPCR) has been proposed to effectively detect viral particles, and it does not involve Ct values or standard curves. In this study, we designed specific primers and probes, based on conserved regions of viral genomes, to optimize protocols for a dual ddPCR assay for detecting BCoV and BRV and a multiplex ddPCR assay for BEV, BCoV, and BRV. Sensitivity assays revealed that the lower limit of detection for qPCR was 1,000 copies/μL and for ddPCR for BEV, BCoV, and BRV, 2.7 copies/μL, 1 copy/μL and 2.4 copies/μL, respectively. Studying 82 samples collected from diarrheal calves on a farm, our dual ddPCR method detected BCoV, BRV, and co-infection at rates of 18.29%, 14.63%, and 6.1%, respectively. In contrast, conventional qPCR methods detected BCoV, BRV, and co-infection at rates of 10.98%, 12.2%, and 3.66%, respectively. On the other hand, studying 68 samples from another farm, qPCR detected BCoV, BRV, BEV, and co-infection of BCoV and BEV at rates of 14.49%, 1.45%, 5.80%, and 1.45%, respectively. Our multiplex ddPCR method detected BCoV, BRV, BEV, co-infection of BCoV and BEV, and co-infection of BRV and BEV. at rates of 14.49%, 2.9%, 8.7%, 2.9%, and 1.45%, respectively. Studying 93 samples from another farm, qPCR detected BCoV, BRV, BEV, and co-infection of BCoV and BEV was detected at rates of 5.38%, 1.08%, 18.28%, and 1.08%, respectively. Co-infection of BCoV, BRV, BEV, BCoV, and BEV, and co-infection of BRV and BEV, were detected by multiplex ddPCR methods at rates of 5.38%, 2.15%, 20.45%, 1.08%, and 1.08%, respectively. These results indicated that our optimized dual and multiplex ddPCR methods were more effective than conventional qPCR assays to detect these viral infections.

Keywords: bovine coronavirus; bovine enterovirus; bovine rotavirus; droplet digital PCR; real-time quantitative PCR.