基于CRISPR/Cas12a的叶酸代谢位点MTHFR基因C677T多态性检测方法的建立
Establishment of adetection method for MTHFR gene C677T polymorphism in folate metabolism using CRISPR/Cas12a
田石 1安莉莎 2马瑶 1金孝华 2马旭 2张璐2
作者信息
- 1. 北京市海淀区妇幼保健院,北京 100080
- 2. 国家卫生健康委科学技术研究所,北京 100081;国家人类遗传资源中心,北京 100024
- 折叠
摘要
目的 开发一种基于CRISPR/Cas12a技术的叶酸代谢位点c.677(MTHFR C677T)的高效检测方法,以实现对C677T多态性的快速、准确分析.方法 采用重组酶聚合酶扩增(RPA)结合CRISPR/Cas12a建立单管检测反应体系,建立人MTHFR基因C677T基因分型策略;测试200例孕妇人群样本MTHFR基因C677T多态性,并与常规PCR产物测序结果进行一致率比较.结果 基于CRISPR/Cas12a检测人MTHFR基因C677T多态性的检测方法结果准确、特异性好,与PCR产物测序结果具有高度一致性.结论 建立的基于CRISPR/Cas12a检测技术的人MTHFR C677T基因分型方法简单、快捷、精准,为叶酸代谢位点MTHFR C677T基因型检测提供了新的途径,具有潜在的临床应用前景.
Abstract
Objective:To develop an efficient detection method based on CRISPR/Cas12a technology for the folate metabolism site c.677(MTHFR C677T),enabling rapid and accurate analysis of the C677T polymorphism.Methods:A single-tube detection system was established by combining recombinase polymerase amplification(RPA)with CRISPR/Cas12a to develop a genotyping strategy for the human MTHFR gene C677T polymorphism.Two hundred pregnant woman samples were tested for MTHFR gene C677T polymorphism,and the results were compared for concordance with conventional polymerase chain reaction(PCR)product sequencing.Results:The detection method for the human MTHFR gene C677T polymorphism,utilizing the CRISPR/Cas12a system,demonstrated high accuracy and excellent specificity.The results exhibited a remarkably high concordance with those obtained from PCR product sequencing.Conclusions:The established genotyping method for the human MTHFR C677T gene,utilizing CRISPR/Cas12a detection technology,is simple,rapid,and precise.This method provides a novel approach for the genotyping of the folate metabolism site c.677,holding promising potential for clinical applications.
关键词
人亚甲基四氢叶酸还原酶基因/多态性/CRISPR/Cas12a/恒温扩增Key words
Human methylene tetrahydrofolate reductase gene/Polymorphism/CRISPR/Cas12a/Isothermal amplification引用本文复制引用
基金项目
国家自然科学基金(82200261)
海淀区属卫生健康系统高层次人才发展计划(2022HDXD006)
出版年
2024
国家科技期刊平台
NSTL
万方数据