Establishment and Preliminary Application of a Real-time RT-qPCR Assay for the Detection of Bovine Enterovirus

Purpose The aim of this study was to establish a real-time RT-qPCR detection method for bovine enteroviruses (BEV), and to investigate the prevalence of BEV infection in calves in Kunming region, providing a reference for the diagnosis, prevention, and vaccine development of BEV.

Methods Primers for conventional RT-PCR and RT-qPCR were designed based on the 5' UTR and 3D region of BEV gene. Positive samples were amplified via RT-PCR to prepare recombinant plasmids. The RT-qPCR detection method was established by optimizing reaction conditions, constructing standard and melting curves, and evaluating specificity and sensitivity. Using this method, 219 diarrheic calf samples from 15 intensive cattle farms in Kunming were tested to analyze BEV infection prevalence.

Results The assay exhibited excellent linearity within the range of 13.6×10²-13.6×10⁹ copies/μL, with a standard curve of y=−3.725x+39.571 (R²=0.9998). Only BEV showed specific amplification when tested alongside other bovine diarrheal viruses, with a detection limit of 13.6×10¹ copies/μL. The intra-assay and inter-assay coefficients of variation for Ct values were 0.23%-0.59% and 0.81%-1.78%, respectively, both less than 2%, indicating that the method is simple to operate, highly specific, highly sensitive, and highly stable. The positive detection rate in Kunming was 12.33% (27/219), with infection rates of 4.44%, 10.13%, and 17.89% in calves aged <1 week, 1 week to 2 months, and 2-6 months, respectively, showing an age-dependent increase. Phylogenetic analysis revealed that the prevalent BEV strains in Kunming were genotypes E and F, closely related to multiple domestic strains.

Conclusion This study establishes a real-time RT-qPCR detection method for BEV, providing a rapid, sensitive, and specific technical approach for the diagnosis and epidemiological investigation of this virus.