Drop-off微滴式数字PCR检测急性髓系白血病NRAS基因突变及其临床应用

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Abstract Objective： To establish a new dropoff droplet digital PCR (ddPCR) method for detecting NRAS mutations in patients with acute myeloid leukemia (AML), and to evaluate its clinical value. Methods： Specialized primers and probes were designed for NRAS G12 and G13 mutation. This research established and optimized the reaction system of drop-off ddPCR and verified this method. A total of 140 clinical samples were screened by this method, and the results were compared with Sanger sequencing, and verified by next generation sequencing (NGS). Results： Dropoff droplet digital PCR method was established to detect different types of the G12 and G13 mutations of NRAS in AML patients. The method had high sensitivity (up to 0.158%), good repeatability and linearity. The limit of blank was 5.40 copies/μL and the limit of detection was 15.84 copies/μL. Among 140 newly diagnosed AML patients, 28 samples (20%) were detected by dropoff ddPCR, the mutation rate was from 0.26%-52.85% (median 2.14%); only 7 (5%) samples were detected positive by Sanger sequencing. For further verification, those samples which were detected negatively by Sanger but positively by drop-off ddPCR were sent for NGS. A total of 14 samples were detected positive by NGS, the mutant frequency of the residual 7 detected positive by dropoff ddPCR was 0.53%-1.50% (median 1.10%). Conclusion： Drop-off ddPCR can effectively detect the quantity of the G12 and G13 mutations of NRAS, which can be used for screening and judging the prognosis of AML patients.

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Received: 02 August 2022

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http://zzs.ujs.edu.cn/xbyxb/EN/ OR http://zzs.ujs.edu.cn/xbyxb/EN/Y2023/V33/I01/49

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