The incidence of prostate cancer (PCa) is increasing at the highest rate among male cancers, and early diagnosis is essential for timely intervention and improved prognosis.DNA methylation is the earliest molecular marker of tumors, which is an ideal target for early diagnosis. In particular, methylation liquid biopsy of circulating tumor DNA (ctDNA) in body fluids such as blood and urine is expected to achieve early and convenient PCa diagnosis. However, existing PCR or high-throughput sequencing methods are not optimal for methylation clinical detection due to poor accuracy or high cost. Consequently, it is necessary to develop more accurate, convenient, and low-cost methylation liquid biopsy methods to achieve earlier clinical diagnosis of PCa.

First, a PCa-specific methylation diagnostic panel was constructed using bioinformatics methods. Then, a one-pot signal sensing platform was constructed based on the photoactivated amplification system combined with the CRISPR-Cas12a system for direct detection of bisulfite-converted methylated DNA. Following which, probe compositions targeting multiple PCa methylation sites were designed, and the detection platform was validated and optimized in synthetic and PCa cell line DNAs, respectively. Finally, blood and urine samples from PCa and healthy controls from Tongji Hospital were collected to conduct clinical trials for diagnosis employing the photoactivated one-pot detection platform.

Using simulated methylated DNA, we verified the photoactivated CRISPR-Cas signal sensing platform possesses good signal amplification and can accurately identify trace methylation sites submerged in normal DNA with a discriminatory degree of 302. A PCa-specific methylation sites were screened in the DNA methylomics database and a 6-gene diagnostic panel for PCa was constructed, including GSTP1, HOXA7, ADCY4, AOX1, RASSF5, and SFRP5. Methylation-specific PCR probe systems were constructed for each methylation site, and the method was validated in cell lines to sensitively distinguish PCa methylated DNA with a detection limit of down to 0.005%. On this basis, a one-pot assay for the rapid detection of tumor methylated DNA was constructed by combining with RPA thermostatic amplification technology, which can shorten the detection time to 2.5 hours. In 55 body fluid (blood or urine) samples from PCa patients and healthy volunteers, the method showed a sensitivity of 89% and a specificity of 85%.

Altogether, we here constructed a novel photoactivatable DNA methylation detection system, bridging the gap between high-sensitivity and low-cost, which is difficult to balance in conventional methods. The system, combined with a specific methylation diagnostic panel and thermostatic amplification technology, can realize rapid and precise diagnosis of PCa, and is of great clinical promotion significance in PCa liquid biopsy.