Development and validation of a reverse transcription-insulated isothermal PCR assay for detection of caprine kobuvirus and its epidemiological and molecular characterization in China.

Abstract

Caprine kobuvirus (CKoV) is an emerging diarrheal pathogen in goats. We developed and validated a reverse transcription-insulated isothermal PCR (iiPCR) targeting the conserved 3D gene for detection of CKoV. The assay demonstrated high analytical specificity, a limit of detection of 39.9 copies·μL^-1, and good repeatability. A total of 722 goat fecal samples were collected from five farms in Sichuan, Chongqing, Yunnan, and Hunan in China and tested using the newly developed method. The overall CKoV positivity rate was 40.44% (292/722), and the farm-level positivity rate was 100% (5/5), with the highest rate in Hunan (84.00%, 21/25), followed by Yunnan (42.86%, 48/112), Sichuan (38.58%, 206/534), and Chongqing (33.33%, 17/51). Lambs exhibited substantially higher positivity rates (57.14-83.33%) than goats aged ≥3 months (13.39-15.52%), and chi-square test confirmed a statistically significant association between age and CKoV positivity (χ ² = 252.97, df = 6, p < 0.001), confirming age-dependent susceptibility in lambs. Twelve full-length VP0 and VP1 sequences were obtained (GenBank: PZ424409-PZ424432). Phylogenetic analysis revealed that all isolates formed an independent clade related to known CKoV strains. The VP0 sequences shared 97.28-100% amino acid identity, while the VP1 sequences shared 98.61-100% identity. Compared with the reference strain (GenBank: MT584793.1), stable substitutions were found in the VP0 receptor-binding domain and a conserved VP1 region, potentially affecting protein conformation or function. This study provides a validated detection tool and new insights into CKoV epidemiology, genetic diversity, and evolution.