Fig. 1: CreDiT assay for HPV detection.
a Streamlined onsite workflow. The assay executes the nucleic acid (NA) extraction and the CreDiT reaction in a single pot, minimizing the hands-on times. The total assay time is about 36 min. b CreDiT assay principle. Two reactions concurrently occur: recombinase polymerase amplification (RPA) and Cas12a-based signal generation. RPA is the major reaction that replicates the target DNA. During RPA, the polymerase displaces the DNA duplex, exposing single-stranded regions recognizable by Cas12a/gRNA complexes. This recognition event activates the complexes to cleave dye-quencher (F-Q) probes, generating fluorescent signals. As the RPA cycles proceed, more Cas12a/gRNA complexes are activated, amplifying the overall signal. All reactions are carried out at a constant temperature (42 °C) without requiring external interruptions. c A compact, integrated assay system was developed. The device had a turret-shaped heating block accommodating 12 samples and an optical module for fluorescent detection (LED light-emitting diode, PD photodiode). The heating block, mounted on a motor, rotated to position each sample for fluorescent measurements. The device had a small form factor (14 × 11 × 6.5 cm3) and connected to a computer. Custom-designed software running on a computer presented a graphical user interface for device control and real-time data analysis. d Two microcontroller units (MCUs) automated the CreDiT assay processes. MCU1 controlled heating elements to maintain the desired temperatures and rotated the heating block during optical detection. MCU2 generated waveforms for fluorescent excitation and detected the resulting signal.
