Zulauf Brian, Pastey Manoj K
Department of Veterinary Biomedical Sciences, Oregon State University, Corvallis, OR, United States.
Front Vet Sci. 2025 Aug 29;12:1645647. doi: 10.3389/fvets.2025.1645647. eCollection 2025.
Bovine respiratory syncytial virus (BRSV) and bovine parainfluenza virus Type 3 (BPIV3) are ubiquitous respiratory pathogens of cattle, contributing significantly to the bovine respiratory disease (BRD) complex. Rapid and reliable detection methods are essential to mitigate economic losses and improve animal welfare. This study aimed to develop and validate sensitive and specific reverse transcriptase quantitative real-time PCR (RT-qPCR) assays for the simultaneous detection of BRSV and BPIV3 in bovine respiratory samples. Primers and dual-labeled probes were designed from GenBank sequences targeting conserved regions of the BRSV N gene and BPIV3 NP gene and optimized for sensitivity and specificity. The assays were evaluated using reference strains, field isolates, and clinical samples. Analytical sensitivity was established through serial dilutions of transcribed RNA and confirmed by probit regression analysis, yielding LOD95 values of 164 genome copies for BRSV and 359 genome copies for BPIV3. The assays demonstrated high specificity (no cross-reactivity with non-target bovine respiratory viruses), and reproducibility (CV < 5%). Standard curves demonstrated strong linearity (R > 0.99) with amplification efficiencies of 104.2% for BRSV and 81.6% for BPIV3. Diagnostic performance was evaluated on 100 clinical samples, with monoplex RT-qPCR detecting BRSV in 19% and BPIV3 in 11% of cases, outperforming virus isolation. The multiplex assay detected 17% BRSV and 7% BPIV3 positives of cases in a single reaction. Compared to traditional virus isolation, the RT-qPCR assays detected 2.4 × more BRSV and reliably identified BPIV3-positive cases that were otherwise missed. These assays offer a robust diagnostic solution for high-throughput screening in clinical and surveillance settings.
牛呼吸道合胞病毒（BRSV）和牛副流感病毒3型（BPIV3）是牛群中普遍存在的呼吸道病原体，对牛呼吸道疾病（BRD）复合体有重大影响。快速可靠的检测方法对于减少经济损失和改善动物福利至关重要。本研究旨在开发并验证灵敏且特异的逆转录定量实时PCR（RT-qPCR）检测方法，用于同时检测牛呼吸道样本中的BRSV和BPIV3。根据GenBank序列，针对BRSV N基因和BPIV3 NP基因的保守区域设计引物和双标记探针，并对其灵敏度和特异性进行优化。使用参考毒株、田间分离株和临床样本对检测方法进行评估。通过对转录RNA进行系列稀释确定分析灵敏度，并通过概率回归分析进行确认，得出BRSV的LOD95值为164个基因组拷贝，BPIV3为359个基因组拷贝。该检测方法具有高特异性（与非目标牛呼吸道病毒无交叉反应）和可重复性（CV<5%）。标准曲线显示出强线性（R>0.99），BRSV的扩增效率为104.2%，BPIV3为81.6%。对100份临床样本进行诊断性能评估，单重RT-qPCR检测到19%的病例中存在BRSV，11%的病例中存在BPIV3，优于病毒分离法。多重检测在单个反应中检测到17%的BRSV阳性病例和7%的BPIV3阳性病例。与传统病毒分离法相比，RT-qPCR检测方法检测到的BRSV多2.4倍，并可靠地鉴定出原本漏检的BPIV3阳性病例。这些检测方法为临床和监测环境中的高通量筛查提供了强大的诊断解决方案。
