Development and Validation of a High-Resolution Melting (HRM) Method for Differentiating Ovis and Equi Biovars of...

C. pseudotuberculosis is a Gram-positive pathogenic bacterium that infects various animals, causing diseases such as caseous lymphadenitis, leading to significant economic losses in the livestock industry and posing zoonotic risks. This study targeted the conserved gyrA gene fragment of this bacterium, designed specific primers, optimized the reaction system and conditions, and established a high-resolution melting curve (HRM) detection method with potential utility for preliminary molecular screening. Validation showed that this method exhibits strong specificity, producing specific amplification only for the target biovars. The melting temperatures (Tm values) for the two biovars were 86.16 ± 0.05 °C and 86.92 ± 0.05 °C, respectively, allowing clear differentiation. It demonstrated high sensitivity, with minimum detection limits of 28 copies/μL and 25 copies/μL for standard plasmids of the ovis and equi biovars, respectively. The method also showed good reproducibility, with intra- and inter-batch coefficients of variation both below 1.0%. Applied to 133 clinical nasal swab samples from goats in Fujian Province, the method detected a positivity rate of 19.5% and indicates that a biovar equi-like gene fragment was detected in goat nasal swabs from Fujian Province via molecular screening. The HRM method developed in this study is sensitive, specific, simple, and cost-effective, enabling rapid detection and biovar differentiation of C. pseudotuberculosis. It is suitable for large-scale clinical sample screening and provides an efficient technical approach for epidemiological monitoring and precise control of the disease.