#0330

Development and Validation of a Novel Technique for Precise Diagnosis of DNA Mutations in Prostate Cancer

X. Zhong¹, Y. Xiong¹, S. Wang¹, Q. Xia¹, Y. Yang¹

¹Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Department of Urology, Wuhan, China

Introduction:

The rising incidence of prostate cancer (PCa) underscores the need for accurate diagnostic and monitoring techniques to inform treatment decisions and enhance patient outcomes. DNA mutations play a pivotal role in PCa progression and are critical for therapeutic guidance. Traditional detection methods, such as PCR and high-throughput sequencing, face challenges of low sensitivity and high costs, highlighting the urgent need for convenient, sensitive, and cost-effective mutation detection methods for PCa precise diagnosis and monitoring.

Material and methods:

A DNA mutation detection system was developed that eliminates purification and single-stranded conversion, utilizing the ME I and CRISPR-Cas12a systems. Bioinformatics were employed to analyze PCa mutation sites and design specific fluorescent probes. Mutation detection was performed across PCa tissues to establish a standard curve. Then, we created a streamlined platform for DNA extraction, amplification, and detection by integrating the ME I/CRISPR-Cas12a system with a thermostatic amplification method. Finally, samples from 56 PCa patients with DNA mutations were collected for clinical diagnostic.

Results:

The ME I/CRISPR-Cas12a system demonstrated direct detection of mutated DNA with a discriminatory factor of 278.4. The method successfully identified eight mutation types with a discriminatory factor reaching 184. We designed and validated probe systems targeting 3 PCa mutation sites (ATM, BRCA2, CHEK2) in mimic samples, achieving discrimination factors between 154 and 310. The integrated platform, combined with thermostatic amplification, showed a detection limit of less than 0.01% for BRCA2 mutations, with an assay cost of approximately $9.50 and a processing time of about 30 minutes—50% shorter than traditional methods. The platform effectively detected BRCA2 mutations in 56 PCa patients, achieving 100% sensitivity, with the detected mutation abundance in line with the high-throughput sequencing results.