| 基于国产微滴数字PCR的SNP快速检测技术研究 |
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| 引用本文： | 林培双,王寒,祝令香,贝蕾,黄江,赵雯婷,李彩霞.基于国产微滴数字PCR的SNP快速检测技术研究[J].生物化学与生物物理进展,2022,49(6):1123-1134. |
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| 作者姓名： | 林培双 王寒 祝令香 贝蕾 黄江 赵雯婷 李彩霞 |
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| 作者单位： | 1）贵州医科大学法医学院，贵阳 550004,2）公安部物证鉴定中心，法医遗传学公安部重点实验室，现场物证溯源技术国家工程实验室，北京 100038,3）新羿制造科技有限公司，北京 100038,3）新羿制造科技有限公司，北京 100038,1）贵州医科大学法医学院，贵阳 550004,2）公安部物证鉴定中心，法医遗传学公安部重点实验室，现场物证溯源技术国家工程实验室，北京 100038,1）贵州医科大学法医学院，贵阳 550004;2）公安部物证鉴定中心，法医遗传学公安部重点实验室，现场物证溯源技术国家工程实验室，北京 100038 |
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| 基金项目： | 国家自然科学基金（81772027）资助项目。 |
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| 摘 要： | 目的 在法医学领域,现有的SNP检测主要依赖进口,检测工作量大、耗时长且成本较高。微滴数字PCR (droplet digital PCR,ddPCR)作为新一代的PCR技术,可以快速检测低浓度样本DNA,且有较强的抗干扰能力。本研究旨在国产ddPCR平台建立SNP分型检测体系并对其性能进行评估,以探讨ddPCR技术在法医学检验领域的应用价值。方法 在ddPCR平台建立高原适应性EPAS1单倍型(rs115321619、rs73926263、rs73926264、rs73926265和rs55981512)检测体系,测试各位点引物探针特异性,对体系的准确性、稳定性、灵敏度、检材适应性进行评估,并比较了ddPCR和SNaPshot微测序检测体系的抗抑制性,最后对样本地区来源进行测试。结果 ddPCR在2.5 h内即可快速获取检测结果,体系准确性和稳定性好,检测灵敏度为0.312 5 ng,且抗抑制性能力突出。70份测试样本检测结果与背景信息一致。结论 基于ddPCR的SNP检测体系具有准确可靠、简便快速、抗抑制能力强等优势,在法医学快速检验领域有较强的应用潜力,适合法医现场检验需求。
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| 关 键 词： | 微滴数字PCR 法医学检验 SNP分型 |
| 收稿时间： | 2021/11/3 0:00:00 |
| 修稿时间： | 2022/2/18 0:00:00 |
Rapid Genotyping of SNP Based on Domestic Droplet Digital PCR |
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| LIN Pei-Shuang,WANG Han,ZHU Ling-Xiang,BEI Lei,HUANG Jiang,ZHAO Wen-Ting and LI Cai-Xia.Rapid Genotyping of SNP Based on Domestic Droplet Digital PCR[J].Progress In Biochemistry and Biophysics,2022,49(6):1123-1134. |
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| Authors: | LIN Pei-Shuang WANG Han ZHU Ling-Xiang BEI Lei HUANG Jiang ZHAO Wen-Ting and LI Cai-Xia |
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| Institution: | 1)Institute of Forensic Medicine, Guizhou Medical University, Guiyang 550004, China,2)Key Laboratory of Forensic Genetics, Beijing Engineering Research Center of Crime Scene Evidence Examination, National Engineering Laboratory for Forensic Science, Institute of Forensic Science, Beijing 100038, China,3)TargetingOne Corporation, Beijing 100038, China,3)TargetingOne Corporation, Beijing 100038, China,1)Institute of Forensic Medicine, Guizhou Medical University, Guiyang 550004, China,2)Key Laboratory of Forensic Genetics, Beijing Engineering Research Center of Crime Scene Evidence Examination, National Engineering Laboratory for Forensic Science, Institute of Forensic Science, Beijing 100038, China,1)Institute of Forensic Medicine, Guizhou Medical University, Guiyang 550004, China;2)Key Laboratory of Forensic Genetics, Beijing Engineering Research Center of Crime Scene Evidence Examination, National Engineering Laboratory for Forensic Science, Institute of Forensic Science, Beijing 100038, China |
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| Abstract: | Objective Current forensic SNP genotyping methods often require imported platforms and are labor-intensive, time-consuming and costly. Droplet digital PCR (ddPCR) is a new generation of PCR technology that allows rapid qualification of rare target DNA sequence, and is less susceptible to PCR inhibitors. This study is intended to establish a SNP genotyping method on domestic ddPCR platform and explore the applicability of ddPCR in forensics.Methods Genotyping system of the high altitude adaptive EPAS1 haplotype (rs115321619, rs73926263, rs73926264, rs73926265 and rs55981512) was established by ddPCR. Then we tested the specificity of primers and probes, evaluated its accuracy, stability, sensitivity and adaptability separately. Meanwhile, ddPCR and SNaPshot minisequencing technology were compared in terms of inhibitor resistance ability. Finally, preliminary application in 70 samples were conducted.Results The ddPCR assay only required a total run time within 2.5 h, and showed high accuracy and repeatability in SNP genotyping. The detection sensitivity was 0.312 5 ng. DdPCR assays also exhibited better tolerance to inhibitors than SNaPshot. The tested individuals showed good consistence with their background information.Conclusion The SNP genotyping assay based on ddPCR is accurate, rapid, easily-used and shows great resistance to perturbations by inhibitors, which has strong application potential in the field of rapid forensic detection, and is suitable for forensic analysis. |
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| Keywords: | droplet digital PCR forensic detection SNP genotyping |
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