A single-tube dual-channel CRISPR platform for genotyping of waterfowl reoviruses.

Waterfowl reovirus (WRVs) includes two genotypes, Genotype I (Muscovy duck reovirus, MDRV) and Genotype II (novel duck reovirus, NDRV). They have the same clinical manifestations, but different pathogenicity, and can co-infect individual hosts, resulting in diagnostic ambiguity that cannot be solved by single-target detection. Here, we report a single-tube dual-channel platform coupling recombinase polymerase amplification (RPA) with orthogonal CRISPR effectors: LbCas12a targeting the conserved S4 gene of MDRV via a ROX-labeled ssDNA reporter, and LwaCas13a targeting the S1 gene of NDRV via a FAM-labeled ssRNA reporter. The integrated format enables simultaneous genotypic differentiation within 30 min at isothermal temperatures, producing exclusive ROX-channel signal for MDRV, exclusive FAM-channel signal for NDRV, and concurrent dual-channel signal for co-infected samples, with no cross-channel interference (p > 0.05). The assay achieved a limit of detection of 1 copy/μL for both targets with no cross-reactivity against four co-circulating waterfowl pathogens. An ImageJ-based RGB quantification standard enables equipment-minimal visual classification of all four diagnostic outcomes. Clinical evaluation of 133 field specimens yielded complete concordance (100%) with qPCR, correctly identifying 15 MDRV-positive, 67 NDRV-positive, 8 co-infected, and 43 negative cases. This platform provides a validated, equipment-minimal tool for concurrent WRV genotyping and field surveillance.