Establishment of a PCR-CRISPR/Cas12a-based visual detection method for DNMT3A R882H/C

JIANG Qingyang; SUN Rumei; MIU Xiaping; FAN Xijie; ZHANG Haoming; XIA Yinxiao; LI Kaimin; CHEN Tao

doi:10.12122/j.issn.1674-4500.2025.05.09

Abstract：Objective To develop a DNMT3A R882H(c.2645G>A)and R882C(c.2644C>T)visual detection system based on polymerase chain reaction(PCR)and clustered regularly interspaced short palindromic repeats(CRISPR)with their associated protein(Cas12a).Methods Thirty-four bone marrow samples from patients suspected of acute myeloid leukemia were collected at a hospital from September 2023 to September 2024.The specific amplification primers and CRISPR RNA(crRNA)for the PCR-Cas12a nucleic acid detection system were designed to target the DNMT3A R882H/C mutation sites.Primer pairs with optimal amplification performance were selected using agarose gel electrophoresis,and the fluorescence intensity generated by Cas12a protein cleavage of nearby fluorescent probes was used to identify the crRNA with the highest cleavage efficiency,based on the signal difference between mutant and wild-type sequences,to construct the PCR-Cas12a Nucleic Acid Detection System.Wild-type human genomic DNA and DNMT3A R882H/C mutant plasmids were mixed in varying ratios to simulate different mutation frequencies and assess the detection limit of PCR-Cas12a.Finally,clinical samples validated for DNMT3A R882H/C mutations by high-throughput sequencing were tested using PCR-Cas12a to evaluate methodological consistency and resistance to interference.Results The PCR-Cas12a nucleic acid visual detection system effectively detected DNMT3A R882H/C mutations in clinical samples with mutation frequencies as low as 1%.Conclusion This study established a visual detection method for DNMT3A R882H/C mutations by combining PCR with CRISPR-Cas12a technology,which is rapid,specific,and sensitive,providing robust support for the prognostic assessment of acute myeloid leukemia.