tetano
Editor, Senior Moderator
Front Cell Infect Microbiol
. 2026 Sep 16:16:1922968.
doi: 10.3389/fcimb.2026.1922968. eCollection 2026.
Zhenghan Luo # 1 2 , Minzhi Xu # 1 , Letian Zhang 1 , Yifang Han 1 , Anqi Tan 1 , Taiwu Wang 1 , Fuqiang Ye 1 , Chunhui Wang 1 , Jinhai Zhang 1
Affiliations
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.
Keywords: CRISPR/Cas12a; ERA-CRISPR/Cas12a; H5; H7; novel influenza A virus; reverse transcription enzymatic recombinase amplification (RT-ERA).
. 2026 Sep 16:16:1922968.
doi: 10.3389/fcimb.2026.1922968. eCollection 2026.
Establishment and preliminary evaluation of an RT-ERA combined with CRISPR/Cas12a assay for detection of H5 and H7 influenza viruses
Zhenghan Luo # 1 2 , Minzhi Xu # 1 , Letian Zhang 1 , Yifang Han 1 , Anqi Tan 1 , Taiwu Wang 1 , Fuqiang Ye 1 , Chunhui Wang 1 , Jinhai Zhang 1
Affiliations
- PMID: 42818682
- PMCID: PMC13623710
- DOI: 10.3389/fcimb.2026.1922968
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.
Keywords: CRISPR/Cas12a; ERA-CRISPR/Cas12a; H5; H7; novel influenza A virus; reverse transcription enzymatic recombinase amplification (RT-ERA).