A One-Pot CRISPR-Cas12b Assay for Rapid Detection of Human Adenovirus Serotypes 3 and 7

Human adenovirus (HAdV) is a leading cause of acute respiratory tract infections (ARTIs) in children. The high prevalence of HAdV serotypes 3 and 7 in regions such as China presents a significant public health challenge. Here, we propose a one-pot assay that integrates multiple cross displacement amplification (MCDA) with CRISPR-Cas12b for the detection of HAdV-3 and HAdV-7, termed HAdV-MCDA-One. In this system, MCDA provides exponential target amplification, while the collateral cleavage activity of Cas12b enables secondary signal amplification. The entire reaction is performed isothermally at 60°C in a single tube, providing a fluorescent readout within 50 min, making the assay suitable for point-of-care testing (POCT). Leveraging the single-base recognition capability of CRISPR-Cas12b, the assay demonstrates high specificity, with no cross-reactivity observed against the other 13 identified pathogens. The limit of detection was determined to be 1.59 copies per reaction using target plasmids. Moreover, when evaluated with 96 clinical pharyngeal swabs, the assay showed 100% concordance with quantitative PCR (qPCR), confirming its clinical reliability. These results demonstrate HAdV-MCDA-One as a rapid and robust tool for HAdV-3 and HAdV-7 detection, with significant potential for clinical diagnosis and public health surveillance.