One-pot synthesized three-way junction based multiple strand displacement amplification for sensitive assay of H5N1 DNA†

Abstract

The rapid and sensitive detection of H5N1, a highly pathogenic avian influenza virus, is crucial for controlling its spread and minimizing its impact on public health. In this study, we developed a novel biosensor based on strand displacement amplification (SDA) coupled with CRISPR/Cas12a for highly sensitive detection of H5N1 DNA. The biosensor utilizes a combination of a three-way junction structure, composed of three hairpins (H1, H2, H3), to initiate amplification through SDA, resulting in the production of numerous activators. These activators then trigger CRISPR/Cas12a's collateral cleavage activity, which generates a detectable fluorescence signal. The biosensor demonstrated a linear detection range from 100 fM to 800 pM, with a detection limit as low as 72.87 fM. The optimized biosensor exhibited excellent sensitivity, high specificity, and a broad dynamic range, making it a promising tool for the early detection of H5N1 DNA in complex biological samples. Additionally, the use of CRISPR/Cas12a's trans-cleavage activity significantly improved signal amplification and specificity, allowing for more reliable detection compared to traditional methods. The results highlight the advantages of the integrated SDA and CRISPR/Cas12a approach, which addresses the limitations of conventional detection methods, such as low sensitivity, lengthy analysis times, and high costs. The biosensor's ability to perform well in complex sample matrices demonstrates its potential for point-of-care diagnostics, especially in resource-limited settings. Future applications of this technology could extend to the detection of other pathogens, offering a versatile and adaptable platform for disease surveillance and management.