Fluorescent primers amplification refractory mutation system qPCR (FP ARMS-qPCR) for MTHFR C677T SNP genotyping.

IF 2.6 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Molecular Biology Reports Pub Date : 2024-11-06 DOI:10.1007/s11033-024-10036-3

Yongjun Han, Hong Wu

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引用次数: 0

Abstract

Background: The currently used methods for the detection of methylene tetrahydrofolic acid reductase (MTHFR) C677T single nucleotide polymorphism (SNP) are either time-consuming or expensive. In this study, we devised an accurate, rapid and easy-to-use SNP detection system based on fluorescent primers amplification refractory mutation system qPCR, known as FP ARMS-qPCR.

Methods: Fluorescent primers (FPs) modified by fluorescent dye or quencher near the 3' terminal thymine were designed. The reaction conditions were optimized and the performance was evaluated. Using commercial kits as controls, 242 samples were tested in parallel to verify the feasibility of the FP ARMS-qPCR assay.

Results: We demonstrated the good sensitivity and specificity of the FP ARMS-qPCR with optimized conditions. The assay was able to accurately distinguish between different SNP sites of MTHFR C677T in less than 2 h using as low as 50 pg of template genomic DNA. Completely consistent genotyping results reveal that FP ARMS-qPCR is concordant with commercial kits.

Conclusion: We established a specific, sensitive, and rapid FP ARMS-qPCR method for the detection of MTHFR C677T genotype. This could also serve as a potential diagnostic tool for a variety of diseases.

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用于 MTHFR C677T SNP 基因分型的荧光引物扩增难治突变系统 qPCR (FP ARMS-qPCR)。

背景：目前用于检测亚甲基四氢叶酸还原酶（MTHFR）C677T单核苷酸多态性（SNP）的方法要么费时，要么昂贵。在这项研究中，我们设计了一种基于荧光引物扩增难治性突变系统 qPCR 的准确、快速、易用的 SNP 检测系统，即 FP ARMS-qPCR：方法：设计了在 3' 末端胸腺嘧啶附近用荧光染料或淬灭剂修饰的荧光引物（FPs）。对反应条件进行了优化，并对性能进行了评估。以商业试剂盒为对照，对 242 份样本进行了平行测试，以验证 FP ARMS-qPCR 分析的可行性：结果：在优化条件下，我们证明了 FP ARMS-qPCR 具有良好的灵敏度和特异性。在不到 2 小时的时间内，该检测方法就能准确区分 MTHFR C677T 的不同 SNP 位点，使用的模板基因组 DNA 低至 50 pg。完全一致的基因分型结果表明，FP ARMS-qPCR 与商业试剂盒的结果一致：我们建立了一种特异、灵敏、快速的 FP ARMS-qPCR 方法来检测 MTHFR C677T 基因型。结论：我们建立了特异、灵敏、快速的 FP ARMS-qPCR 方法来检测 MTHFR C677T 基因型，这也可作为多种疾病的潜在诊断工具。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Molecular Biology Reports 生物-生化与分子生物学

CiteScore

5.00

自引率

0.00%

发文量

1048

审稿时长

5.6 months

期刊介绍： Molecular Biology Reports publishes original research papers and review articles that demonstrate novel molecular and cellular findings in both eukaryotes (animals, plants, algae, funghi) and prokaryotes (bacteria and archaea).The journal publishes results of both fundamental and translational research as well as new techniques that advance experimental progress in the field and presents original research papers, short communications and (mini-) reviews.