Dual‐Mode CRISPR/Cas13a Assay for the Detection of Human Metapneumovirus in Clinical Respiratory Samples

Human metapneumovirus (HMPV) is a significant pathogen, causing widespread acute respiratory infections (ARIs). Rapid and accurate detection is crucial for timely diagnosis and outbreak control. To address the limitations of real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR)-based HMPV detection, we developed a novel method integrating recombinase-aided amplification (RAA) with CRISPR-Cas13a technology. Based on the alignment of 335 HMPV whole-genome sequences, a conserved region of the nucleocapsid (N) gene was selected for RAA primer and CRISPR RNA (crRNA) design. The concentrations of Cas protein, crRNA, and probe were optimized for specificity, sensitivity, and repeatability. A dual-mode platform was established, combining a fluorescence assay for quantitative detection and a lateral flow assay (LFA) for visual, on-site readout. Both methods were validated using 106 clinical samples and compared with RT-qPCR. Both methods demonstrated high specificity (no cross-reactivity with other respiratory viruses), a detection limit of 1 copy/μL, and excellent repeatability. Clinical validation showed complete concordance (κ = 1, p < 0.001) between the fluorescence assay and RT-qPCR, and high agreement for LFA (κ = 0.924, p < 0.001), with a sensitivity of 94.23%, specificity of 98.15%, positive predictive value (PPV) of 98%, and negative predictive value (NPV) of 94.6%. This study presents an RAA-CRISPR/Cas13a dual-mode platform for HMPV detection, providing a robust molecular diagnostic framework that paves the way for the future development of rapid testing assays.