Development of Real-Time Fluorescence CRISPR/Cas12a-Based Detection as a Portable Diagnostic System Using Integrated Circuits

Abstract

A solution for achieving high-performance measurements in a space-constrained experimental setup was developed as a portable incubating instrument for real-time fluorescence detection of AvrPi9 gene in rice blast fungus by using a calibrated spectrometer in CRISPR-Cas12a detection. The system demonstrates accurate temperature control with low energy consumption and low deviation of ±0.16 °C from the setpoint temperatures, with high sensitivity and accurate detection within 10 min. The CRISPR-Cas12a detection reaction was demonstrated using AvrPi9 PCR product, crRNAs, LbCas12a and fluorescence-quencher reporter incubating at 37 °C for 10 min. Calibrated C12666MA spectrometer with 480 nm and 520 nm LEDs vs HR4000 reference exhibits low RMS of 0.54 and 1.30 and drift of 6.4 nm and 4.84 nm, respectively indicating high accuracy and reliability in fluorescence detection. Fluorescence signals were observed under an LED transilluminator, while real-time analysis was conducted through spectrometric measurements upon excitation by a 480 nm high-intensity blue LED source. Accuracy of detection between positive, non-template and non-target control was reported with no incidence of false positives observed. The instrument exhibits reliable quantitative detection capabilities with a limit of detection of 3.8 ng of DNA targets that are comparable to when running the same reaction on a commercial real-time PCR, with a detection limit of 1 ng. This study demonstrates that the CRISPR-Cas12a detection method represents a significant breakthrough in molecular diagnostics due to its advantages of rapidity, high sensitivity, and convenience allowing for the development of a compact, and energy-efficient platform that can facilitate real-time on-site diagnostics with accurate temperature control.