LwaCas13a protein- and crRNA-enabled RPA-CRISPR/Cas13a platform for rapid visual differentiation of goose parvovirus and novel goose parvovirus.

Abstract

Waterfowl parvoviruses, including goose parvovirus and novel goose parvovirus, are highly transmissible pathogens that pose serious threats to flock health and the poultry industry. In this paper, we report a macromolecule-enabled recombinase polymerase amplification (RPA)-CRISPR/Cas13a platform for the rapid, sensitive, and field-deployable visual detection of these viruses by, targeting VP3. Guided by crRNA, LwaCas13a specifically recognizes complementary target RNA and is activated to exert non-specific collateral cleavage activity, efficiently degrading nearby single-stranded RNAs. Species-conserved VP3 fragments with stable interspecies differences were used to design RPA primers and crRNAs, and reaction conditions were systematically optimized. This dual-mode system integrates real-time fluorescence with lateral flow test strips detection, achieving fluorescence detection limits of 1 × 100 copies/μL and lateral flow test strips detection limits of 1 × 101 copies/μL for goose parvovirus and 1 × 102copies/μL for novel goose parvovirus, with no cross-reactivity with common avian pathogens. Clinical validation demonstrated high concordance with qPCR and superior sensitivity to conventional PCR. Overall, this macromolecule-driven RPA-CRISPR/Cas13a assay offers a robust, rapid, and accurate tool for the on-site differential diagnosis of waterfowl parvoviruses, enabling timely surveillance and disease control.