Establishment and preliminary evaluation of an RT-ERA combined with CRISPR/Cas12a assay for detection of H5 and H7 influenza viruses

Abstract

Background/objectives Novel influenza A viruses (NIAV) infection is a zoonotic acute respiratory disease. The H5N1 and H7N9 subtypes cause high mortality, posing major public health threats. This study aimed to establish rapid, field-deployable subtype-specific nucleic acid detection assays for H5 and H7 by integrating reverse-transcription enzymatic recombinase amplification (RT-ERA) with CRISPR/Cas12a. Two separate reactions are required per sample. Methods Conserved hemagglutinin (H) sequences were retrieved from the NCBI database, and primers, probes, and crRNAs were designed using Primer Premier 5 and Primer-BLAST. The sensitivity of RT-ERA alone was compared with that of the ERA-CRISPR/Cas12a using serially diluted virus strain nucleic acid. The specificity was verified against other respiratory pathogens with similar clinical manifestations. Results The optimal primers were F1R2 for H5 and F2R3 for H7. The limit of detection (LOD) of RT-ERA alone was 2.63×10³ copies/µL for H5 and 1.86×10³ copies/µL for H7; the ERA-CRISPR/Cas12a assay improved the LOD to 2.63×10¹ copies/µL for H5 and 1.86 copies/µL for H7, representing 100-fold and 1000-fold enhancements in sensitivity, respectively. Specificity testing showed that the assay exclusively detected H5N1 and H7N9 with no cross-reactivity to other tested pathogens. Conclusion The ERA-CRISPR/Cas12a method exhibits enhanced sensitivity for H5 and H7 subtypes detection, along with high specificity, rapidity, and minimal equipment requirements. It offers a promising screening tool for NIAV infections.