Li Qingqing, Pan Yan, Wu Cuilan, Ma Chunxia, Li Jun, Li Changting, Bai Huili, Gong Yu, Liu Jing, Tao Li, Yin Yangyan, Teng Ling, Zhong Shuhong, Lan Meiyi, Hu Shuai, Xuan Xiongbiao, Wei Tianchao, Peng Hao
College of Animal Science and Technology, Guangxi University, Nanning 530004, China.
Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, Nanning 530001, China.
Transbound Emerg Dis. 2024 Feb 10;2024:7108960. doi: 10.1155/2024/7108960. eCollection 2024.
Bovine coronavirus (BCoV) is a notable pathogen affecting newly born calves and adult cattle, increasing mortality rates among calves and reducing productivity in meat and dairy industries, thereby causing substantial economic losses. Current primary laboratory methods for detecting BCoV include RT-PCR assay, real-time RT-PCR assay, and ELISA. However, these methods are time-consuming, require specialized technicians, and necessitate a laboratory environment. Consequently, there is an urgent need for a rapid, sensitive, and easy to use diagnostic method to detect BCoV. This study introduces two innovative protocols: the real-time fluorescent reverse transcription recombinase-aided amplification (RT-RAA) and the test strip RT-RAA (RT-RAA-LFD). Our results indicate that real-time RT-RAA can complete the reaction in 20 min at 39°C, while RT-RAA-LFD can achieve detection in just 17.5 min at 35°C. These new approaches offer higher specificity, with no cross-reactivity to other viruses, and significantly enhanced sensitivity compared to existing methods (1.46 × 10 and 1.46 × 10 copies/L, respectively). We evaluated the performance of our methods using 242 clinical samples, and compared with RT-PCR and RT-qPCR. Both real-time RT-RAA and RT-qPCR yielded similar detection rates, the detection rate of RT-RAA-LFD was better than RT-PCR. The RT-RAA methods developed in this study effectively overcome the limitations associated with both RT-PCR and RT-qPCR by offering advantages including a single, low reaction temperature that allows for room temperature operation. Both methods boast shorter reaction times, simpler and more portable instrumentation, as well as reduced technical and environmental demands. Generally, both RT-RAA methods established in this study offer new avenues for the rapid detection of BCoV, contributing significantly to the monitoring, prevention, and control of the disease in global bovine industry.
牛冠状病毒（BCoV）是一种影响新生犊牛和成年牛的重要病原体，会提高犊牛死亡率并降低肉类和乳制品行业的生产力，从而造成重大经济损失。目前检测BCoV的主要实验室方法包括逆转录聚合酶链反应（RT-PCR）检测、实时荧光定量逆转录聚合酶链反应（real-time RT-PCR）检测和酶联免疫吸附测定（ELISA）。然而，这些方法耗时较长，需要专业技术人员，且需要实验室环境。因此，迫切需要一种快速、灵敏且易于使用的诊断方法来检测BCoV。本研究介绍了两种创新方案：实时荧光逆转录重组酶介导的等温扩增技术（RT-RAA）和试纸条RT-RAA（RT-RAA-LFD）。我们的结果表明，实时RT-RAA能在39℃下20分钟内完成反应，而RT-RAA-LFD在35℃下仅需17.5分钟就能实现检测。这些新方法具有更高的特异性，与其他病毒无交叉反应，并且与现有方法相比灵敏度显著提高（分别为1.46×10和1.46×10拷贝/升）。我们使用242份临床样本评估了这些方法的性能，并与RT-PCR和实时荧光定量PCR（RT-qPCR）进行比较。实时RT-RAA和RT-qPCR的检测率相似，RT-RAA-LFD的检测率优于RT-PCR。本研究开发的RT-RAA方法有效克服了RT-PCR和RT-qPCR的局限性，具有单一、低温反应的优势，可在室温下操作。两种方法都具有反应时间短、仪器更简单便携以及对技术和环境要求降低等优点。总体而言，本研究建立的两种RT-RAA方法为BCoV的快速检测提供了新途径，对全球养牛业中该疾病的监测、预防和控制具有重要意义。
