Visual and Equipment-Minimized Detection of Avian Metapneumovirus Subtype C by RT-RPA-CRISPR/Cas12a Coupled with Lateral Flow Dipstick

Abstract

Avian metapneumovirus (aMPV) is the main etiological agent of turkey rhinotracheitis, causing respiratory distress and economic losses in turkeys. While its impact in broilers and laying hens is relatively limited, aMPV subtype C (aMPV/C) is an important pathogen in ducks and waterfowl, leading to respiratory distress and severe egg-drop syndrome. To overcome the limitations of current diagnostics, such as heavy reliance on laboratory infrastructure and prolonged turnaround times, we established a rapid detection assay integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a and a lateral flow dipstick (LFD), targeting the conserved N gene of aMPV/C. Reaction parameters were systematically optimized, with 30 nmol/L Cas12a and 40 nmol/L crRNA identified as the optimal concentrations. Rigorous specificity evaluation against seven common avian pathogens revealed no cross-reactivity. The limit of detection (LOD) was determined to be as low as 1.3 × 101 copies/μL, outperforming conventional PCR. Validation using clinical samples demonstrated a 98.85% concordance rate with quantitative PCR (qPCR). This platform enables visual detection of aMPV/C within 1 h with minimal equipment requirements. Combining high sensitivity, absolute specificity, and operational simplicity, this assay shows promise as a practical tool for point-of-care diagnostics and epidemiological surveillance of aMPV/C.