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

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。

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.