Multienzyme isothermal rapid amplification combined with CRISPR/Cas12a for sensitive prostate cancer screening using urine collected without digital rectal examination

Abstract

Prostate cancer associated 3 (PCA3), a specific nucleic acid biomarker of prostate cancer, may appear earlier than the prostate-specific antigen (PSA) in prostate cancer. At present, urine, to be used for PCA3 detection, is collected after digital rectal examination (DRE). However, omitting DRE without negatively affecting assay sensitivity may help standardize urine collection procedures and increase PCA3 diagnostic applicability. Based on the DNA replication function of multienzyme isothermal rapid amplification (MIRA) and the specific DNA-shearing function of CRISPR/Cas12a, the MIRA-CRISPR/Cas12a “one-pot” system was developed. The system was shown to detect 0.01 ng/µL RNA in 40 min at room temperature. In addition, this “one-pot” ambient temperature reaction system reduced aerosol contamination and reliance on expensive equipment. Of note, the assay results could be obtained with the naked eye under ultraviolet light. Finally, PCA3 detection results revealed that the marker had great potential for prostate cancer screening, with a specificity of 83.3 %. Compared with the 33.3 % specificity of serum PSA, the specificity of PCA3 for prostate cancer screening was increased by approximately 50 %.