Comparative Evaluation of 16S rRNA Target Regions Using PCR High-Resolution Melt Curve Analysis for Differentiation of Bacteria Associated with Bovine Mastitis.

Bovine bacterial mastitis remains a major challenge for the dairy industry, and timely identification of causative bacteria is essential for effective disease management. This study compared six regions of the 16S rRNA gene for PCR high-resolution melt (HRM) curve analysis to identify the most suitable target for species-level differentiation of mastitis-associated bacteria. A total of 547 clinical bovine milk samples from cows with clinical mastitis were cultured, and representative isolates of 18 bacterial species were selected for comparative analysis. Species identity was confirmed using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) prior to molecular analysis. All six primer pair combinations were tested across the 18 species panel, and discriminatory performance was assessed using a genotype confidence percentage (GCP)-based classification model that allowed for objective interpretation without visual inspection. Among the six target regions, the 203 bp amplicon generated by primer pair F1R1, spanning the V1-to-V2 region and part of V3, showed the strongest overall discriminatory performance with no cross-classification observed. Sequencing of F1R1 amplicons confirmed species identity and showed concordance with HRM-based classification. Phylogenetic analysis of sequences from this region showed clustering broadly consistent with established taxonomic relationships. Proof-of-concept testing showed that the workflow could also be applied to DNA extracted directly from selected milk samples without prior bacterial isolation. Overall, this approach provides a rapid, cost-effective, and high-throughput method for preliminary discrimination of mastitis-associated bacteria.