Analysis for Bopivirus B in goats in the Sichuan province, China using a novel TaqMan real-time polymerase chain reaction assay.

Abstract

Background: Bopivirus B is an emerging picornavirus that affects goats in China. This study aimed to establish a TaqMan real-time PCR assay for detecting Bopivirus B and conduct a preliminary survey of infection in six goat farms in Sichuan province, China. Specific primers and a probe targeting the 3D gene of Bopivirus B were designed, and the TaqMan-based real-time PCR assay was successfully established following the optimization of reaction conditions and components. A total of 257 goat fecal samples were collected from six farms in Sichuan and tested using the newly developed method.

Results: The assay demonstrated a linear relationship between 2.73 × 10³ and 2.73 × 10⁹ copies/µL, with a high correlation coefficient (R² = 0.999) and amplification efficiency of 109%. Additionally, the assay exhibited excellent specificity and reproducibility, with a detection limit of 27.3 copies/µL. The field positive rate of Bopivirus B was 100%, and a higher positive rate was observed in diarrheal fecal samples (33.72%) compared to non-diarrheal fecal samples (12.28%, P < 0.005), suggesting a potential association between Bopivirus B and goat diarrhea, with a widespread prevalence in goats in the Sichuan province. Furthermore, ten complete 3D genes sequences of Bopivirus B were obtained, and phylogenetic analysis showed that all Bopivirus B strains in this study were most closely related to two known Chinese Bopivirus strains based on nucleotide sequences of the 3D gene.

Conclusions: This study developed a highly specific, repeatable, and sensitive TaqMan-based real-time PCR assay targeting the 3D gene for Bopivirus B detection, offering a valuable tool for the detection and epidemiological investigation of Bopivirus B. The prevalence of Bopivirus B was widespread in goats in China, with a close association observed between Bopivirus B and goat diarrhea.