1Tangshan Academy of Agricultural Sciences, Tangshan063000, China
2Tangshan Center for Animal Disease Control and Prevention, Tangshan063000, China
3Tangshan Animal Quarantine Station, Tangshan063000, China
4Tangshan Normal University, Tangshan063000, China
*Corresponding Author: Lixue Dong, Tangshan Center for Animal Disease Control and Prevention, Tangshan063000, China, Email: wangxiaoyue821@163.com
**Corresponding Author: Zhiyong Liu, Tangshan Center for Animal Disease Control and Prevention, Tangshan063000, China, Email: tslzy2005@163.com
#These authors contributed equally to this work.
The aim of this study was to establish a TaqMan-MGB multiplex real-time fluorescent quantitative PCR assay for the simultaneous detection of four pathogens, enabling rapid and effective identification of the etiological agents of bovine mastitis.
Specific primers and TaqMan-MGB probes were designed targeting the conserved gene sequences of Staphylococcus aureus, Escherichia coli, Salmonella spp and Pseudomonas aeruginosa. After optimizing reaction conditions including primer/ probe concentrations and annealing temperature, a multiplex real-time PCR assay was established. Specificity, sensitivity and repeatability tests were conducted and the established method was applied to detect 192 clinical samples.
The amplification efficiencies of the 4-plex real-time PCR assay were all above 90%, with correlation coefficients R2 exceeding 0.99, indicating high amplification efficiency. The assay specifically amplified S. aureus, E. coli, Salmonella spp and P. aeruginosa, with no cross-reactivity with Bacillus cereus, Klebsiella spp, or Listeria spp, demonstrating strong specificity. The assay exhibited high sensitivity, with the minimum detection limits of 1.85×101 copise/μL for S. aureus, 2.02×101 copise/μL for E. coli, 1.93×101 copise/μL for Salmonella spp and 1.89×101 copise/μL for P. aeruginosa. Repeatability tests revealed intra-assay coefficients of variation (CV) less than 1.86% and inter-assay CV less than 2.68%, confirming good reproducibility. When applied to 192 clinical milk samples, the coincidence rate of the 4-plex assay with conventional single-plex PCR for detecting mixed infections with the four bacteria was 83.30% and the overall coincidence rate between the two methods was 95.70%. The established 4-plex real-time PCR assay provides a valuable tool for the detection of bovine mastitis pathogens, guiding clinical medication and facilitating molecular epidemiological studies.
Bovine mastitis, Detection method, Multiplex real-time fluorescent quantitative PCR, TaqMan-MGB
