Abstract
Background:
Bovine mastitis caused by Streptococcus agalactiae (GBS) and Klebsiella pneumoniae (KP) causes severe economic losses in the dairy industry. The rapid, accurate, and on-site detection of these pathogens is essential for timely diagnosis and control.

Aim:
This study aimed to develop and validate a duplex recombinase polymerase amplification (RPA) assay for the simultaneous, rapid, and sensitive detection of GBS and KP in bovine mastitis.

Methods:
Conserved cfb (GBS) and rcsA (KP) genes were targeted, and specific primer pairs were designed for duplex RPA assay development. The reaction conditions, including amplification temperature, time, and primer ratios, were optimized. Analytical sensitivity was assessed using serial dilutions of genomic DNA, and specificity was evaluated against 6 non-target bacterial species. Clinical applicability was determined by testing 35 milk samples from cows with mastitis using duplex RPA and conventional PCR.

Results:
The optimized duplex RPA assay was completed within 30 min at 39 °C within 30 minutes with balanced primer concentrations, enabling the simultaneous detection of GBS and KP. The assay exhibited high specificity with no cross-reactions. The detection limit was 10-5 ng/μL, exceeding the sensitivity of PCR (10-3 ng/μL). Among the clinical samples, duplex RPA identified 14.3% GBS-positive, 8.6% KP-positive, and 8.6% co–infected cases. The overall PCR concordance was 97.1%, with a Kappa coefficient of >0.75.

Conclusion:
Duplex RPA enabled the rapid, specific, and sensitive detection of GBS and KP in bovine mastitis, with superior analytical sensitivity compared with PCR.

Key words: Bovine mastitis; Streptococcus agalactiae; Klebsiella pneumoniae; RPA; Duplex detection.