Multiplex one-step RT–qPCR assays for simultaneous detection of BRV, BCoV, Escherichia coli K99+ and Cryptosporidium parvum

作者：Xinru Zhao, Min Li, Yingying Yang, Yidan Wang, Xiaoru Zheng, Dehua Yin, Haihui Gao, Huatao Li, Kaiqiang Fu, Zhi Cao · 发表于：Frontiers in Veterinary Science · 年份：2025 · DOI：10.3389/fvets.2025.1561533 · 被引用次数：6 · 研究领域：Animal health and immunology、Microbial infections and disease research、Molecular Biology Techniques and Applications

Introduction Bovine rotavirus (BRV), bovine coronavirus (BCoV), Escherichia coli K99 + ( E. coli K99 + ), and Cryptosporidium parvum ( C. parvum ) are the most common pathogens involved in calf production. These pathogens can cause calf diarrhea, leading to significant economic losses in the cattle farming industry. These four pathogens have similar clinical symptoms, making them difficult to distinguish. Therefore, we established a one-step quadruple Taq Man fluorescence quantitative PCR method capable of simultaneously and rapidly detecting BRV, BCoV, E. coli K99 + , and C. parvum . Methods Specific primers and Taq Man probes were designed for the BRV VP-6 gene, BCoV N gene, E. coli K99 + K99 gene, and C. parvum 18S rRNA gene. Standard positive plasmids were constructed, and the reaction conditions of the method were optimized. The sensitivity, specificity, and repeatability of the method were validated, and clinical samples were tested. Results The minimum detection limits of this method for BRV, BCoV, E. coli K99 + , and C. parvum were 5.8 × 10 1 , 2.3 × 10 1 , 4.5 × 10 2 , and 2.6 × 10 1 copies/μL, respectively. The intra- and intergroup coefficients of variation were all less than 1.2%. This method has the advantages of strong specificity, reproducibility, low cost, and no cross-reaction with other bovine pathogens. Compared with the commercial reagent kit method were used to analyze clinical samples, and both the diagnostic sensitivity (DSe) and diagnostic specificity ...