Rapid field visual detection of avian metapneumovirus by integrating MIRA pre-amplification with CRISPR-Cas13a to enhance sensitivity and specificity: Innovative technologies well-suited for real-time large-scale epidemiological surveillance.

Avian metapnemovirus (aMPV) is associated with swollen head syndrome in poultry and causes significant economic losses due to respiratory disease and reduced egg production, hence the urgent need for rapid and reliable detection methods. To address this challenge, we developed two novel CRISPR-Cas13a combined multiplex isothermal recombinase amplification (MIRA) assays: a fluorescence-based method (Fluorescent-MIRA-Cas13a) and a lateral flow strip-based platform (LF-MIRA-Cas13a), representing the first application of CRISPR-Cas13a for aMPV detection. Through systematic screening of 21 primer pairs and 4 crRNAs, optimal combinations were identified, achieving excellent specificity with no cross-reactivity against 12 common poultry pathogens. The assays demonstrated high sensitivities of 2 copies/reaction (Fluorescent-MIRA-Cas13a) and 5 copies/reaction (LF-MIRA-Cas13a), outperforming qPCR (20 copies/reaction), while maintaining reproducibility across inter- and intra-assay tests. Clinical evaluation using 200 suspected samples revealed 21 % (Fluorescent-MIRA-Cas13a) and 17.5 % (LF-MIRA-Cas13a) positivity rates, aligning closely with qPCR (19 %) and showing strong diagnostic concordance (κ > 0.93). Notably, both methods enabled detection within 1 h, without requiring specialized plug-in instruments (only a handheld UV emitter or lateral flow strips), significantly reducing operational complexity compared to conventional techniques. These field-deployable assays provide a cost-effective solution for rapid on-site diagnosis and large-scale epidemiological surveillance, particularly in resource-limited settings.