Establishment of a multiplex qPCR assay for the detection of pathogens associated with bovine respiratory disease complex

作者：Linghao Li, Qifeng Jiang, Siying Li, Siying Li, Xin Li, Sheng Sun, Xiyi Wang, Chuangqi Sun, Kun Jia, Shoujun Li, Shoujun Li · 发表于：Frontiers in Veterinary Science · 年份：2025 · DOI：10.3389/fvets.2025.1594488 · 被引用次数：9 · 研究领域：Microbial infections and disease research、Respiratory viral infections research、Animal Disease Management and Epidemiology

Introduction The bovine respiratory disease complex poses a significant threat to the cattle industry, necessitating a multifaceted approach to address its occurrence. The syndrome is caused by various pathogens such as bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus type 3 (BPIV3), bovine viral diarrhea virus (BVDV), bovine adenovirus type 3 (BAV3), Mycoplasma bovis (Mb), and infectious bovine rhinotracheitis virus (IBRV). The confluence of these pathogens causes substantial economic losses to the cattle industry. Although preventive and control measures have been implemented, containment of bovine respiratory diseases continues to present a formidable challenge, highlighting the need for innovative diagnostic and intervention strategies. Methods In this study, we designed specific primers targeting six conserved pathogen genes ( N of BRSV, M of BPIV3, 5’UTR of BVDV, Hexon of BAV3, oppF of Mb, and gB of IBRV). Subsequently, we established a multiplexed fluorescent real-time quantitative PCR (qPCR) assay for simultaneous detection of these pathogens. Results The developed method exhibited high specificity and sensitivity, with the lowest detection limits for plasmid DNA standards of BRSV, BPIV3, BVDV, BAV3, Mb, and IBRV being 70.1, 40.4, 15.1, 74.4, 69.6, and 4.99 copies/μL, respectively. The coefficients of variation determined by the assay established in this study were <4%, and the amplification efficiency was 93.84%–111.60%, which showed the reliabil...