A rapid and ultrasensitive RPA-assisted CRISPR–Cas12a/Cas13a nucleic acid diagnostic platform with a smartphone-based portable device

The spread of infectious diseases can be controlled by early identification of the source of infection and timely diagnosis to stop transmission. Real-time fluorescence quantitative polymerase chain reaction (PCR) is the current gold standard for pathogen diagnosis, with high detection sensitivity and accuracy. However, due to the need for specialized equipment, laboratories, and personnel, it is difficult to achieve rapid and immediate diagnosis during large-scale infectious disease outbreaks. Herein, an optimized CRISPR-based nucleic acid detection method was developed that reduces the CRISPR detection time to 15 min while maintaining high sensitivity. By using nucleic acid extraction-free and lyophilization techniques, the ‘sample-in-result-out’ detection of the two target genes of SARS-CoV-2, the human internal reference gene, and the negative quality control sample can be completed in 20 min, with a sensitivity of 0.5 copies/μL. Additionally, to facilitate the application, a smartphone-based reverse transcription-recombinase polymerase amplification (RT-RPA)-assisted CRISPR-rapid, portable nucleic acid detection device was developed, integrating functions such as heating, centrifugation, mixing, optical detection and result output. Process control, output, and uploading of detection results were conducted through smartphones. The device is not dependent on a power supply and can perform on-site rapid virus detection in resource-limited settings. Real-time uploading of results helps to rapidly implement epidemic prevention and control measures, providing an innovative means of detection, control, and prevention of virus-based infectious diseases. This important work provides a new and effective tool to manage potential future outbreaks of infectious diseases.