[A CRISPR-Cas13a-based amplification-free electrochemical biosensor for rapid detection of bovine viral diarrhea...

Bovine viral diarrhea virus (BVDV), a major pathogen in the global bovine industry, causes diarrhea, fever, and reproductive disorders, leading to substantial economic losses. Developing the methods for rapid and accurate detection of BVDV is crucial for epidemic control. Current detection methods have notable limitations. PCR-based nucleic acid amplification techniques rely on sophisticated instruments and complex procedures. CRISPR-Cas13a systems, despite their high specificity, still require nucleic acid pre-amplification, which results in cumbersome workflows and contamination risks. To establish a simpler and more efficient on-site detection method for BVDV, this study integrated the CRISPR-Cas13a system characterized by specific recognition with electrochemical sensing praised for efficient signal transduction to establish a novel nucleic acid amplification-free method for the detection of BVDV. Through optimization of key parameters, including CRISPR RNA (crRNA) combination, buffer components, and Cas13a/crRNA concentration ratio, the biosensor achieved a detection limit of 3 090 copies/μL-representing a 4-5 order of magnitude improvement in sensitivity compared with conventional Cas13a fluorescence-based detection-and completed the entire process from sample loading to result output within 35 min. Specificity tests demonstrated that the sensor exclusively detected BVDV without cross-reactivity to other common bovine viruses (bovine parainfluenza virus type 3, bovine respiratory syncytial virus, bluetongue virus, and foot-and-mouth disease virus). Clinical validation with 22 samples demonstrated 100% specificity and sensitivity. The developed CRISPR-Cas13a-based electrochemical biosensor offers the advantages of being nucleic acid amplification-free and operationally simple, serving as a powerful new tool for rapid on-site BVDV detection with significant potential for veterinary diagnostics and epidemic prevention and control.