CRISPR technology has recently emerged as a powerful biosensing tool for sensitive and specific nucleic acid detection when coupled with isothermal amplification (e.g., recombinase polymerase amplification (RPA)). However, it remains a challenge to incorporate isothermal amplification into CRISPR detection in a one-pot system due to their poor compatibility. Here, we developed a simple CRISPR gel biosensing platform for human immunodeficiency virus (HIV) RNA detection by combining reverse transcription-recombinase polymerase amplification (RT-RPA) reaction solution with a CRISPR gel. In our CRISPR gel biosensing platform, CRISPR-Cas12a enzymes are embedded into the agarose gel, providing a spatially separated but connected reaction interface with the RT-RPA reaction solution. During isothermal incubation, the RT-RPA amplification occurs initially on the CRISPR gel. When RPA products are sufficiently amplified and reach the CRISPR gel, the CRISPR reaction occurs in the whole tube. With the CRISPR gel biosensing platform, we successfully detected down to 30 copies of HIV RNA per test within 30 min. Furthermore, we validated its clinical utility by detecting HIV clinical plasma samples, achieving superior performance compared with the real-time RT-PCR method. Thus, our one-pot CRISPR gel biosensing platform demonstrates great potential for rapid and sensitive molecular detection of HIV and other pathogens at the point of care.
Keywords: Agarose gel; CRISPR-Cas12a; Human immunodeficiency virus; Point-of-care diagnostics; Recombinase polymerase amplification.
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