ArticleVeterinary research communications2026
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.
Article in Veterinary research communications, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
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.
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