Rapid, highly specific and sensitive detection of human adenovirus using multiple cross displacement amplification coupled with CRISPR-Cas12a-based detection

Human adenoviruses (HAdV), particularly serotypes 3 and 7, are significant pathogens that cause respiratory tract infection. Here, we developed a novel HAdV diagnostic assay, termed HAdV-MCDA-CRISPR, by combining multiple cross displacement amplification (MCDA) with CRISPR/Cas12a-based detection. HAdV-MCDA-CRISPR assay uses MCDA to amplify target DNA, followed by detection of predefined sequences using a CRISPR/Cas12a-crRNA complex. Resulting fluorescence from probe cleavage indicates a positive result. A set of 10 MCDA primers and a specific crRNA were designed to ensure high specificity of HAdV-MCDA-CRISPR assay. The entire procedure, including rapid template preparation (∼15 min), MCDA reaction (40 min), and result interpretation (5 min), takes approximately one hour. Targeting the conserved hexon gene, HAdV-MCDA-CRISPR achieves high sensitivity under optimized MCDA reaction conditions (40 min at 66 °C), detecting as little as 1.92 copies/μL of hexon-containing plasmid DNA without cross-reactivity. Validation using 128 clinical samples demonstrated 78/80 positive results for HAdV-positive patients and 48/48 negative results for HAdV-negative patients. Thus, HAdV-MCDA-CRISPR assay offers a promising alternative for rapid and accurate clinical diagnosis of HAdV-3 and HAdV-7 infections.