Development and evaluation of a duplex recombinase polymerase amplification assay for rapid detection of Streptococcus agalactiae and Klebsiella pneumoniae in bovine mastitis.
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引用次数: 0
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 polymerase chain reaction (PCR).
Results: The optimized duplex RPA assay was completed within 30 minutes at 39°C 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.
期刊介绍：
Open Veterinary Journal is a peer-reviewed international open access online and printed journal that publishes high-quality original research articles. reviews, short communications and case reports dedicated to all aspects of veterinary sciences and its related subjects. Research areas include the following: Infectious diseases of zoonotic/food-borne importance, applied biochemistry, parasitology, endocrinology, microbiology, immunology, pathology, pharmacology, physiology, epidemiology, molecular biology, immunogenetics, surgery, ophthalmology, dermatology, oncology and animal reproduction. All papers are peer-reviewed. Moreover, with the presence of well-qualified group of international referees, the process of publication will be done meticulously and to the highest standards.
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