Articles Vol. 67 No. 10 31/10/2026

DEVELOPMENT AND PRELIMINARY VALIDATION OF A SYBR GREEN–BASED ALLELE-SPECIFIC REAL-TIME PCR ASSAY FOR DETECTION OF THE DPYD RS55886062 VARIANT

Nguyen Uoc Nguyen, Nguyen Dang Khoa, Nguyen Huu Ngoc Tuan
DOI: 10.52163/yhc.v67i10.6920
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Abstract

Introduction: The rs55886062 (DPYD*13) variant reduces dihydropyrimidine dehydrogenase (DPD) activity and increases the risk of severe fluoropyrimidine toxicity. Objective:
To develop and preliminarily validate a SYBR Green-based ASO real-time PCR assay for detecting rs55886062. Materials and methods: An experimental study was conducted on 23 DNA samples from whole blood of healthy individuals. Control plasmids representing the T/T, T/G, and G/G genotypes were constructed, confirmed by Sanger sequencing, and used for assay optimization. The assay was preliminarily validated based on amplification efficiency, ΔCt values, and comparison with Sanger sequencing results. Results: Control plasmids carrying the T and G alleles were successfully constructed. The optimized conditions included a 10 µL reaction volume, primer concentration of 0.2 µM, and annealing temperature of 60°C. The assay discriminated genotypes with |ΔCt| ≥5.0 for homozygous genotypes and |ΔCt| <5.0 for heterozygous genotypes, with an amplification efficiency of 90–110% and R² ≥0.99. The T/T genotype results for all 23 genomic DNA samples showed 100% concordance with Sanger sequencing. Conclusion: A SYBR Green-based ASO real-time PCR assay for detecting rs55886062 was successfully developed and preliminarily validated.

Keywords
rs55886062; DPYD; ASO real-time PCR; SYBR Green.
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