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Background <p>The tetra-primer amplification refractory mutation system (T-ARMS) PCR assay is a cost-effective and rapid method for SNP genotyping. The <i>methylenetetrahydrofolate reductase</i> (<i>MTHFR</i>) c.1286&#xa0;A &gt; C (rs1801131) polymorphism can influence MTHFR enzyme activity and homocysteine levels. While its independent clinical impact remains a subject of debate, it is frequently screened in thrombophilia panels, particularly in the context of compound heterozygosity.</p> Methods and results <p>This cross-sectional validation study aims to develop and validate a T-ARMS-PCR assay on the <i>MTHFR</i> c.1286&#xa0;A &gt; C mutation. The results were validated using the Kompetitive Allele-Specific PCR (KASP) as the reference method across 30 clinical DNA samples from a thrombophilia-susceptible cohort. The T-ARMS-PCR assay successfully distinguished genotypes with a clear electrophoretic separation and without non-specific amplification. The results exhibited 100% concordance with the KASP assay (95% CI: 88.4–100%; κ: 1.0). The total turnaround time including the reaction and electrophoretic separation was 107.5&#xa0;min.</p> Conclusions <p>T-ARMS-PCR offers a reliable, rapid, and cost-effective alternative for <i>MTHFR</i> c.1286&#xa0;A &gt; C genotyping. This method is particularly suitable for resource-limited clinical settings that require accurate genotyping without the need for high-end sequencing or specialized instrumentation.</p>

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Development and validation of a tetra-primer ARMS-PCR assay for genotyping the MTHFR (rs1801131) c.1286 A > C (p.Glu429Ala) polymorphism: a comparative study with KASP

  • 期刊论文

  • 发表日期：

    2026年5月16日

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  • Amany Alqosaibi,
  • Akram Husain Rehman Syed Rasheed,
  • Huseyin Tombuloglu,
  • Reham Altwayan,
  • Abdulrahman Alhusil,
  • Taghreed Awadh,
  • Mehmet Ozdemir

摘要

Background

The tetra-primer amplification refractory mutation system (T-ARMS) PCR assay is a cost-effective and rapid method for SNP genotyping. The methylenetetrahydrofolate reductase (MTHFR) c.1286 A > C (rs1801131) polymorphism can influence MTHFR enzyme activity and homocysteine levels. While its independent clinical impact remains a subject of debate, it is frequently screened in thrombophilia panels, particularly in the context of compound heterozygosity.

Methods and results

This cross-sectional validation study aims to develop and validate a T-ARMS-PCR assay on the MTHFR c.1286 A > C mutation. The results were validated using the Kompetitive Allele-Specific PCR (KASP) as the reference method across 30 clinical DNA samples from a thrombophilia-susceptible cohort. The T-ARMS-PCR assay successfully distinguished genotypes with a clear electrophoretic separation and without non-specific amplification. The results exhibited 100% concordance with the KASP assay (95% CI: 88.4–100%; κ: 1.0). The total turnaround time including the reaction and electrophoretic separation was 107.5 min.

Conclusions

T-ARMS-PCR offers a reliable, rapid, and cost-effective alternative for MTHFR c.1286 A > C genotyping. This method is particularly suitable for resource-limited clinical settings that require accurate genotyping without the need for high-end sequencing or specialized instrumentation.