A Multiplex CRISPR-Cas12a-Based Hydrogel Microarray Platform for the Simultaneous Detection of Common Adenovirus Types in Clinical Samples

Adenovirus infections are a leading cause of respiratory and gastrointestinal diseases, representing a significant global health challenge. Rapid and accurate detection of adenovirus types is essential for timely diagnosis and effective management. Traditional diagnostic methods, such as PCR, are often time-consuming and require complex laboratory infrastructure, limiting their application in resource-limited settings. In this study, we present a CRISPR/Cas12a-based assay integrated with a hydrogel microarray for the simultaneous detection of six common adenovirus types (1, 2, 3, 4, 7, and 14). After amplification of adenoviral DNA using recombinase polymerase amplification (RPA), the amplified DNA enters the hydrogel, where it activates the Cas12a-crRNA complex trapped within the gel. This activation leads to the cleavage of an ssDNA reporter, generating a fluorescent signal. The use of a hydrogel microarray enables efficient and multiplexed detection of adenovirus types in a single assay. The method demonstrated high sensitivity, with detection limits ranging from 10 to 50 copies/μL across the six adenovirus types. It also showed excellent specificity, with no cross-reactivity observed with other respiratory viruses. Clinical validation with 30 human adenovirus samples revealed 100% specificity and high concordance with qPCR results. This CRISPR/Cas12a-based hydrogel microarray platform offers a rapid, cost-effective, and highly specific diagnostic tool with significant potential for clinical and public health applications.