基于微滴数字PCR检测线粒体DNA低频异质性  

Droplet digital PCR-based detection of low-frequency heterogeneity in mitochondrial DNA

作　　者：漆小琴 王嘉慧 王雅雅 方晨 程凤 张晓梦 李娜[1] 马鲁豫 王艳[1,3] 严江伟[1,2] Qi Xiaoqin;Wang Jiahui;Wang Yaya;Fang Chen;Cheng Feng;Zhang Xiaomeng;Li Na;Ma Luyu;Wang Yan;Yan Jiangwei(School of Forensic Medicine,Shanxi Medical University,Jinzhong 030600;School of Clinical and Basic Medicine,Shandong First Medical University(Institute of Basic Medicine),Jinan,250117;Department of Medical Innovation Research,General Hospital of the Chinese PLAGeneral Hospital,Beijing,100853)

机构地区：[1]山西医科大学法医学院,山西晋中030600 [2]山东第一医科大学临床与基础医学院(基础医学研究所),山东济南250117 [3]中国人民解放军总医院医学创新研究部,北京100853

出　　处：《中国法医学杂志》2022年第6期579-583,共5页Chinese Journal of Forensic Medicine

基　　金：国家自然基金项目(82030058,82002006)。

摘　　要：目的 基于微滴数字PCR(Droplet digital PCR,ddPCR)技术,建立线粒体DNA低频异质性检测方法。方法 针对DNA标准品9947A和HL-60线粒体分型不同的两个SNP位点(nt93和nt16311)以及9947A存在的异质性位点(nt1393、nt3242和nt7861)设计引物和探针,建立ddPCR反应体系,分别检测9947A和HL-60的6种不同混合比例(1:1、1:4、1:9、1:99、1:999、1:999 9)的异质性模型和9947A的异质性位点,并进行测序验证。结果经过验证,所建方法成功检测出9947A的异质性位点,并能检测出异质性比例低至0.01%的位点。结论 本研究建立的基于ddPCR技术的检测方法,可用于线粒体低频异质性分析。Objective To establish a method for low-frequency heterogeneity detection of mitochondrial DNA based on droplet digital PCR(ddPCR). Methods Primers and probes were designed for two SNP loci(nt93 and nt16311) with different mitochondrial typing of standards 9947A and HL-60, as well as heterogeneity loci(nt1393, nt3242 and nt7861)existing in 9947A, and ddPCR reaction system was established. The heteroplasmy model of 9947A and HL-60 with six different mixing ratios(1:1, 1:4, 1:9, 1:99, 1:999,1:999 9) and the heterogeneity loci of 9947A were detected, and the sequencing was validated. Results After verification, the established method successfully detected 9947A heterogeneity loci, and could detect the heterogeneity ratio as low as 0.01 % loci. Conclusion The detection method based on ddPCR established in this study can be used for mitochondrial low-frequency heterogeneity analysis.

关 键 词：ddPCR 线粒体DNA 低频异质性 

分 类 号：D919.2[医药卫生—法医学]