Zhao Xinru, Li Min, Yang Yingying, Wang Yidan, Zheng Xiaoru, Yin Dehua, Gao Haihui, Li Huatao, Fu Kaiqiang, Cao Zhi
College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, China.
Innovus Solarex Biotech Co., Ltd., Qingdao, China.
Front Vet Sci. 2025 Mar 26;12:1561533. doi: 10.3389/fvets.2025.1561533. eCollection 2025.
Bovine rotavirus (BRV), bovine coronavirus (BCoV), K99 ( K99), and () 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 Man fluorescence quantitative PCR method capable of simultaneously and rapidly detecting BRV, BCoV, K99, and .
Specific primers and Man probes were designed for the BRV VP-6 gene, BCoV N gene, K99 K99 gene, and 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.
The minimum detection limits of this method for BRV, BCoV, K99, and were 5.8 × 10, 2.3 × 10, 4.5 × 10, and 2.6 × 10 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 (DSp) were above 90%, with kappa values greater than 0.9.
The one-step multiplex RT-qPCR method developed in this study for detecting BRV, BCoV, K99, and is expected to be an effective tool for the rapid and economical diagnosis and monitoring of diarrhoeal diseases in calves.
牛轮状病毒（BRV）、牛冠状病毒（BCoV）、K99菌毛（K99）和[未提及的病原体]是犊牛生产中最常见的病原体。这些病原体可导致犊牛腹泻，给养牛业造成重大经济损失。这四种病原体具有相似的临床症状，难以区分。因此，我们建立了一种一步四重荧光定量PCR方法，能够同时快速检测BRV、BCoV、K99菌毛和[未提及的病原体]。
针对BRV VP-6基因、BCoV N基因、K99菌毛K99基因和18S rRNA基因设计特异性引物和荧光探针。构建标准阳性质粒，并优化该方法的反应条件。验证了该方法的灵敏度、特异性和重复性，并对临床样本进行检测。
该方法对BRV、BCoV、K99菌毛和[未提及的病原体]的最低检测限分别为5.8×10、2.3×10、4.5×10和2.6×10拷贝/微升。组内和组间变异系数均小于1.2%。该方法具有特异性强、重复性好、成本低且与其他牛病原体无交叉反应的优点。与商业试剂盒方法用于分析临床样本相比，诊断灵敏度（DSe）和诊断特异性（DSp）均高于90%，kappa值大于0.9。
本研究开发的用于检测BRV、BCoV、K99菌毛和[未提及的病原体]的一步多重逆转录荧光定量PCR方法有望成为快速、经济地诊断和监测犊牛腹泻疾病的有效工具。
