Development and evaluation of a single-tube RT-ERA-CRISPR/Cas12a assay for simultaneous detection of enterovirus G and recombinant strains with papain-like cysteine protease gene insertion

Enterovirus G (EV-G) strains carrying the papain-like cysteine protease (PLCP) gene insertion have recently emerged as important pathogens associated with diarrhea in weaned piglets. Herein, a single-tube reverse transcription-enzymatic recombinase amplification coupled with CRISPR/Cas12a (RT-ERA-CRISPR/Cas12a) dual-readout assay was established to concurrently detect pan-EV-G via conserved RNA-dependent RNA polymerase 3D-encoding gene and screen pathogenic PLCP-carrying recombinant strains. After systematic optimization of primer-probe set and reaction parameters, the analytical and clinical performances of the established assay were evaluated. The assay exhibited no cross-reaction with other common porcine diarrhea-associated viruses or closely related members of the Picornaviridae family. Its limits of detection reached 5.19 copies·µL⁻¹ for the 3D-encoding gene and 5.51 copies·µL⁻¹ for the PLCP gene, demonstrating superior sensitivity compared with conventional RT-PCR, RT-qPCR, RPA, and LAMP assays. Testing 96 field fecal samples yielded almost perfect agreement with RT-qPCR (κ = 0.95, concordance = 97.92%) and moderate consistency with conventional RT-PCR (κ = 0.68, concordance = 87.88%), respectively. Unlike multiplex CRISPR-Cas12a platforms that suffer from reporters' interference from Cas12a non-specific cleavage and dual nfo-probe systems prone to oligonucleotide dimerization, this work implemented separated visual readouts: nfo-probe lateral flow strips for 3D-encoding gene amplicons and Cas12a collateral cleavage for PLCP gene amplification products. An 85 ℃ heating step neutralized endonuclease Ⅳ to remove potential CRISPR signal inhibition, realizing unified single-tube detection. This assay is rapid, cost-efficient, and features high specificity and sensitivity. It offers a practical on-farm surveillance tool for virulent PLCP-recombinant EV-G, facilitating prevention and control of swine diarrheal diseases and safeguarding pork production safety.