Study on the detection of prostate cancer using MIRA-CRISPR/Cas12a technology

Background: Prostate cancer (PCa) is the most diagnosed cancer among men globally and a leading cause of cancer-related mortality. However, current conventional prostate diagnostic methods fail to meet the growing demands of clinical practice in terms of speed, simplicity, sensitivity, and specificity. To address these limitations, we established a molecular detection system based on MIRA-CRISPR/Cas12a technology. Using reverse transcription-multienzyme isothermal rapid amplification (RT-MIRA) to amplify minute PCA3-specific fragments in samples, we employ CRISPR/Cas12a to detect the fluorescence signal released by these fragments, enabling the detection of trace PCA3 molecules in urine. Methods: PCA3 standard strain cultivation and total RNA extraction; establishment and optimization of the MIRA amplification system using primers designed for the PCA3 molecular marker; design of crRNA targeting optimal sites within the detection sequence, combined with CRISPR/Cas12a technology to establish the detection system; preliminary validation of the technology's sensitivity and specificity. Results: the MIRA-CRISPR/Cas12a technology was successfully established for visual detection of PCA3 molecules in prostate cancer. Primer and crRNA sequences within the reaction system were determined. The detection sensitivity for PCA3 molecules in urine reached 1 × 10⁰ copies per µL with excellent specificity. Conclusion: the MIRA-CRISPR/Cas12a technology enables specific detection of PCA3 molecules in urine. This technique features high sensitivity, high specificity, visual results, and simple operation. It does not require specialized laboratory UV imaging equipment; results are visible to the naked eye under LED blue light. Following further optimization, it offers a feasible technical solution for rapid molecular screening of prostate cancer.