A minimal transcription template-based amplification-free CRISPR-Cas13a strategy for DNA detection

CRISPR-Cas13a has shown great potential for the rapid and accurate detection of pathogen nucleic acids. However, conventional CRISPR-Cas13a-based assays typically require pre-amplification, which can introduce aerosol contamination and operational complexities. In this study, we developed a Minimalist transcription template-based Amplification-free CRISPR-Cas13a strategy for DNA detection (MAD). This strategy facilitates the release of pathogen DNA and its annealing with primers from nasopharyngeal swab samples in a straightforward manner, followed by T7 transcription and CRISPR-Cas13a detection, completing the entire process within 40 min. MAD eliminates the need for DNA extraction and pre-amplification while maintaining high sensitivity after optimization, allowing for result visualization via lateral flow strips. Furthermore, evaluation of 167 clinical pediatric samples identified 18 positive cases of human adenovirus, demonstrating a 99.4% concordance in detection compared to standard qPCR. We believe that MAD offers new insights into CRISPR-Cas diagnostics and, due to its simplicity, rapidity, and safety, is poised for widespread application in clinical practice.