SYBR Green I-based real time RT-PCR assay for bovine coronavirus detection

Abstract

Bovine coronavirus (BCoV) is associated with three different clinical syndromes: neonatal calf diarrhea syndrome, winter dysentery causing hemorrhagic enteritis in adult cattle, and respiratory syndrome associated with shipping fever; all of which have a negative impact on animal welfare and cause significant economic losses in the meat and dairy industries. Virological laboratory diagnosis relies primarily on serological and molecular methods. This study aimed to develop a SYBR Green I-based real-time PCR assay for BCoV detection by amplifying a fragment of the viral nucleocapsid gene. Parameters of critical PCR reaction components and thermodynamic conditions were optimized. Dissociation curve analysis confirmed the specificity of the amplification curves (Tm = 85ºC ± 1°C) with no evidence of non-specific amplifications. The assay showed high levels of specificity, repeatability and sensitivity, with a detection limit of 10³·⁰ TCID₅₀/mL. Diagnostic sensitivity was evaluated in fecal samples from animals with clinical symptoms compatible with winter dysentery and from animals without clinical symptoms of enteric disease. The new assay proved to be more sensitive than end-point RT-PCR and viral isolation for BCoV diagnosis.