Development of Rapid Isothermal Detection Methods Real-Time Fluorescence and Lateral Flow Reverse Transcription Recombinase-Aided Amplification Assay for Bovine Coronavirus

Abstract

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 × 101 and 1.46 × 102 copies/μL, 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.