Calf diarrhea is a common disease affecting the global cattle industry, with complex etiology. Bovine kobuvirus (BKoV), an emerging enteric virus, is increasingly recognized as one of the causative agents of calf diarrhea, resulting in considerable economic losses worldwide. The lack of efficient and standardized molecular detection tools for BKoV currently hampers early diagnosis and precise control of this disease.
To address this challenge, a research team from the College of Animal Science and Technology at Inner Mongolia Minzu University designed specific primers targeting the conserved regions of the BKoV 5' untranslated region (5'UTR) and 3D gene, successfully establishing a SYBR Green I-based real-time quantitative PCR (qPCR) detection method. The findings were published online in Microbiology China in April 2026 and appeared in the October 2026 print issue.
Results showed that the method produced positive amplification exclusively for BKoV, with no cross-reactivity against other common bovine diarrhea pathogens, including bovine enterovirus (BEV), bovine viral diarrhea virus (BVDV), bovine norovirus (BNoV), bovine parainfluenza virus type 3 (BPIV3), and bovine torovirus (BToV), and demonstrated excellent specificity. In terms of sensitivity, the assay could detect as few as 5.08 × 10 1 copies/μL of plasmid DNA, which is 10 times more sensitive than conventional RT-PCR. Additionally, both intra- and inter-assay coefficients of variation were below 2%, indicating good reproducibility and stability.
"Our qPCR method is simple to operate and time-efficient. It completes the entire detection process within two hours, which makes it highly suitable for rapid screening in laboratories," said Dr. Shubo Wen, corresponding author of the study and Associate Professor at Inner Mongolia Minzu University. "This research not only provides critical technical support for the epidemiological surveillance of BKoV but also lays a foundation for the future development of multiplex detection kits."
To validate its clinical applicability, the team tested 164 fecal samples from diarrheic calves collected across eight regions in the Inner Mongolia Autonomous Region. The qPCR method identified 53 positive samples, yielding a positivity rate of 32.3%, significantly higher than the 15.9% detection rate achieved by conventional PCR, confirming the prevalence of this virus in local cattle herds. Subsequent nested PCR verification and phylogenetic analysis further validated the accuracy of the results, revealing that the detected BKoV strains were genetically close to those previously reported in northern China.
The research team noted that this new method not only enables accurate quantification of viral load but also facilitates early rapid screening. Future efforts will focus on optimizing the technique to develop a multiplex fluorescence quantitative PCR method capable of simultaneously detecting multiple bovine diarrhea pathogens, enabling rapid differential diagnosis of calf diarrhea etiology and guiding precision medication and comprehensive prevention strategies.
This work was supported by the Youth Science and Technology Talent Project of Inner Mongolia Autonomous Region (NJYT23095, NJYT22053), the National Natural Science Foundation of China (32260848), the Key Science and Technology Project of Inner Mongolia Autonomous Region (2025YFDZ0129), the Central Guidance Fund for Local Science and Technology Development (2025ZY0045), the Natural Science Foundation of Inner Mongolia Autonomous Region (2024MS03002, 2022LHQN03009), and the Innovation and Entrepreneurship Project for Returned Overseas Students of Inner Mongolia (2024LXCX002).
Microbiology China
10.13344/j.microbiol.china.251343
Establishment and application of a quantitative fluorescence PCR detection method for bovine kobuvirus
20-Oct-2026