A purification-free one-pot CRISPR/Cas13a assay for detection of porcine epidemic diarrhea virus.

Authors

Xuan Zhang, Zhuhao Pan, Jiafeng Wen, Chen Wang, Chao Tang, Zhonglin Tang

Published in

Analytica chimica acta. Volume 1422. Pages 346109. Nov 08, 2026. Epub Aug 12, 2026.

Abstract

Porcine epidemic diarrhea virus (PEDV) causes up to 80-100% mortality in neonatal piglets, yet field surveillance remains constrained by RT-qPCR's dependence on column-based nucleic acid purification and centralized laboratory infrastructure-a bottleneck especially severe in the inhibitor-rich matrices typical of swine clinical samples. Here we report an integrated sample-to-answer methodology coupling a purification-free thermal lysis step with one-pot RT-RPA-CRISPR/Cas13a chemistry, delivering PEDV detection within 35 min. Reverse transcription, recombinase polymerase amplification, T7 in vitro transcription, and Cas13a collateral cleavage are confined to a single sealed tube, eliminating open-tube transfer and aerosol contamination. Optimized thermal lysis (80°C, 7 min) liberates amplifiable viral RNA directly from crude anal swabs and feces, obviating column purification. The assay attained 10¹ copies/μL analytical sensitivity with high analytical specificity against six non-target porcine pathogens under the tested conditions. Critically, matrix-tolerance profiling showed the CRISPR-based workflow suppressed inhibition to 7.16% (anal swabs) and 11.89% (feces), versus 63.65% and 69.39% for RT-qPCR (P < 0.01), demonstrating markedly superior robustness under authentic matrices. In a double-blind evaluation of 297 clinical specimens, the platform reached 98.65% concordance with national reference standards (Cohen's κ = 0.941), correctly identifying all 37 RT-qPCR-confirmed positives and additionally resolving four low-titer infections missed by RT-qPCR. By converting an inhibitor-sensitive, infrastructure-bound assay into a purification-free, contamination-resistant workflow with validated clinical reliability, this work provides a robust sample-to-answer analytical strategy for PEDV detection in complex clinical matrices.

PMID:
42763168
Bibliographic data and abstract were imported from PubMed on 20 Sep 2026.

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