A low-cost CHA-integrated CRISPR/Cas12a-based test strip platform for on-site gene detection.

Abstract

On-site nucleic acid detection plays a crucial role in disease diagnosis, biosafety monitoring, and food quality control. This study develops a novel nucleic acid detection platform that integrates catalytic hairpin assembly (CHA) with the CRISPR/Cas12a system and utilizes pregnancy test strips (PTS) for result visualization, addressing the limitations of existing nucleic acid detection methods in balancing sensitivity, specificity, and portability with cost and dependence on a cleanroom. The main mechanism involves the following three steps. The presence of target RNA triggers the CHA reaction, generating double-stranded DNA (dsDNA) as an activation unit. Subsequently, this unit activates the CRISPR/Cas12a system to specifically cleave the single-stranded DNA (ssDNA) that has bridged human chorionic gonadotropin (HCG) to a magnetic bead, ultimately releasing HCG that produces a visual result on the PTS. This dual-signal amplification strategy (CHA cycling and Cas12a trans-cleavage) can detect concentrations as low as 10 pM in approximately 50 min, without the need for pre-amplification of the target nucleic acid. This detection system ensures high sensitivity and specificity while effectively avoiding non-specific activation. In practical applications with transgenic maize samples, the detection results are highly consistent with those of real-time quantitative polymerase chain reaction (qPCR), validating its reliability in real-world scenarios. This innovative method offers advantages such as simple operation and low cost, providing an efficient tool for rapid nucleic acid detection while demonstrating broad potential for application in resource-limited settings.