Standardization and application of ARMS TaqMan real‐time PCR for screening of folate metabolism genes in Han Chinese

IF 3 3区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

ELECTROPHORESIS Pub Date : 2024-09-17 DOI:10.1002/elps.202400017

Peipei Deng, Xuan Liu, Yuanjing Li, Huanhuan Li, Bangrong Zhao, Shusong Wang, Jing Ma

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引用次数: 0

Abstract

Folate has antioxidant properties, and low concentration in seminal plasma may be associated with increased DNA damage in sperm. Mutations of the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes, including MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131), and MTRR A66G (rs1801394), can lead to decreased activity of the encoded folate metabolic enzymes, thereby affecting male reproduction. The current SNP detection methods commonly used in clinical practice have some shortcomings, such as long time‐consuming, complex detection steps, or high cost. The purpose of this study was to establish a simple, time‐saving, sensitive, accurate, and easy to clinical popularization method for folate metabolism gene detection. We combined ARMS‐PCR with TaqMan fluorescent probe to establish an ARMS TaqMan real‐time PCR detection method. According to the variation of rs1801131, rs1801133, and rs1801394, two specific primers (one wild type and one mutant) were designed. Mismatched nucleotides were introduced at the penultimate or third position to improve the specificity of the primer. Specific TaqMan probe was introduced to detect PCR products to improve the sensitivity of the method. The results showed that the sensitivity of ARMS TaqMan real‐time PCR in SNP genotyping was 1 ng, and the accuracy was 100%. A total of 249 clinical samples were detected by the established method, and the correlation between three SNPs and semen quality was analyzed. We found that individuals carrying the AG + GG genotype of rs1801394 had a lower risk of abnormal semen quality. In conclusion, we developed a highly sensitive, accurate, rapid, and easy to be popularized method for detecting SNPs of rs1801394, rs1801131, and rs1801133. ARMS TaqMan real‐time PCR is a reliable SNP genotyping method in folate metabolism genes.

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ARMS TaqMan 实时 PCR 在汉族叶酸代谢基因筛查中的标准化和应用

叶酸具有抗氧化作用，精浆中叶酸浓度低可能与精子中 DNA 损伤增加有关。亚甲基四氢叶酸还原酶（MTHFR）和蛋氨酸合成酶还原酶（MTRR）基因突变，包括MTHFR C677T（rs1801133）、MTHFR A1298C（rs1801131）和MTRR A66G（rs1801394），可导致编码的叶酸代谢酶活性降低，从而影响男性生殖。目前临床上常用的 SNP 检测方法存在一些缺点，如耗时长、检测步骤复杂或成本高。本研究旨在建立一种简单、省时、灵敏、准确、易于临床推广的叶酸代谢基因检测方法。我们将 ARMS-PCR 与 TaqMan 荧光探针相结合，建立了 ARMS TaqMan 实时 PCR 检测方法。根据 rs1801131、rs1801133 和 rs1801394 的变异，设计了两种特异引物（一种野生型引物，一种突变型引物）。在引物的倒数第二个或第三个位置引入了不匹配的核苷酸，以提高引物的特异性。引入特异的 TaqMan 探针检测 PCR 产物，以提高该方法的灵敏度。结果表明，ARMS TaqMan 实时 PCR 在 SNP 基因分型中的灵敏度为 1 ng，准确率为 100%。采用该方法共检测了 249 份临床样本，并分析了三个 SNP 与精液质量的相关性。我们发现，携带 rs1801394 AG + GG 基因型的个体精液质量异常的风险较低。总之，我们开发了一种高灵敏度、准确、快速且易于推广的方法来检测 rs1801394、rs1801131 和 rs1801133 的 SNPs。ARMS TaqMan real-time PCR 是一种可靠的叶酸代谢基因 SNP 基因分型方法。

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来源期刊

ELECTROPHORESIS 生物-分析化学

CiteScore

6.30

自引率

13.80%

发文量

244

审稿时长

1.9 months

期刊介绍： ELECTROPHORESIS is an international journal that publishes original manuscripts on all aspects of electrophoresis, and liquid phase separations (e.g., HPLC, micro- and nano-LC, UHPLC, micro- and nano-fluidics, liquid-phase micro-extractions, etc.). Topics include new or improved analytical and preparative methods, sample preparation, development of theory, and innovative applications of electrophoretic and liquid phase separations methods in the study of nucleic acids, proteins, carbohydrates natural products, pharmaceuticals, food analysis, environmental species and other compounds of importance to the life sciences. Papers in the areas of microfluidics and proteomics, which are not limited to electrophoresis-based methods, will also be accepted for publication. Contributions focused on hyphenated and omics techniques are also of interest. Proteomics is within the scope, if related to its fundamentals and new technical approaches. Proteomics applications are only considered in particular cases. Papers describing the application of standard electrophoretic methods will not be considered. Papers on nanoanalysis intended for publication in ELECTROPHORESIS should focus on one or more of the following topics: • Nanoscale electrokinetics and phenomena related to electric double layer and/or confinement in nano-sized geometry • Single cell and subcellular analysis • Nanosensors and ultrasensitive detection aspects (e.g., involving quantum dots, "nanoelectrodes" or nanospray MS) • Nanoscale/nanopore DNA sequencing (next generation sequencing) • Micro- and nanoscale sample preparation • Nanoparticles and cells analyses by dielectrophoresis • Separation-based analysis using nanoparticles, nanotubes and nanowires.