A novel multiplex RT-qPCR assay for simultaneous detection of bovine norovirus, torovirus, and kobuvirus in fecal samples from diarrheic calves

Abstract

Calf diarrhea results in significant economic loss and is caused by a variety of pathogens, including enteric viruses. Many of these viruses, including bovine norovirus (BNoV), bovine torovirus (BToV), and bovine kobuvirus (BKoV), are recognized as the causative agents of diarrhea; however, they remain understudied as major pathogens. We developed a multiplex reverse-transcription quantitative real-time PCR (RT-qPCR) assay for rapid and simple detection of BNoV, BToV, and BKoV. Our method had high sensitivity and specificity, with detection limits of 1 × 102 copies/μL for BNoV, BToV, and BKoV, which is a lower detection limit than conventional RT-PCR for BNoV and BKoV and identical for BToV. We tested fecal samples from 167 diarrheic calves with our multiplex RT-qPCR method. Viral detection was superior to conventional RT-PCR methods in all samples. The diagnostic sensitivity of the multiplex RT-qPCR method (100%) is higher than that of the conventional RT-PCR methods (87%). Our assay can detect BNoV, BToV, and BKoV in calf feces rapidly and with high sensitivity and specificity.

A multiplex real-time fluorescence-based quantitative PCR assay for calf diarrhea viruses

“…Co-infection of these viruses with other viruses or bacteria complicates diagnosis, making targeted prevention and control measures challenging. Presently, different detection methods have been established to detect the above six pathogens; however, there is no rapid simultaneous detection method (Wang et al, 2019;Chen et al, 2023;Han and Han, 2023). In particular, there are few studies on the detection methods related to BNoV, and the virus can cause severe diarrhea in calves, so the prevention and control of BNoV is relatively difficult, which needs us to pay enough attention.…”