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Establishment and Validation of Plasmid-based Reference Materials for CYP2D6*10 rs1065852 and *41 rs1135840 Detection
Jekmal Malau1, Ahsanal Kasasiah1, Aliya Azkia Zahra1
1Department of Pharmacy, Faculty of Health Science, Universitas Singaperbangsa Karawang, Karawang, West Java, 41361, Indonesia.
Current Pharmaceutical Biotechnology
|May 15, 2026
Summary
New plasmid reference materials accurately detect key CYP2D6 gene variants (rs1065852 and rs1135840) using real-time PCR. These stable, validated materials improve pharmacogenomic testing precision and aid clinical drug therapy optimization.
Area of Science:
- Pharmacogenomics
- Molecular Diagnostics
- Biotechnology
Background:
- The CYP2D6 gene is crucial for drug metabolism and highly polymorphic.
- Specific variants like rs1065852 (CYP2D6*10) and rs1135840 (CYP2D6*41) reduce enzyme activity, impacting drug efficacy.
- Accurate detection of these Single-Nucleotide Polymorphisms (SNPs) is vital for personalized pharmacotherapy.
Purpose of the Study:
- To develop and validate plasmid-derived reference materials for precise real-time PCR genotyping of CYP2D6 SNPs rs1065852 and rs1135840.
- To ensure the analytical performance, stability, and cross-platform compatibility of these novel reference materials.
Main Methods:
- Constructed recombinant plasmids with wild-type and mutant sequences for target SNPs in E. coli.
- Verified sequence integrity using PCR and Sanger sequencing.
- Assessed analytical performance including linearity, Limit of Detection (LoD), allelic discrimination, homogeneity, genetic stability (15 generations), and storage stability (180 days).
- Evaluated cross-platform compatibility across different SNP genotyping assays and real-time PCR instruments.
Main Results:
- Confirmed successful integration of SNPs into plasmids via PCR and sequencing.
- Achieved strong linearity (R² ≥ 0.9874) and a low LoD (10³ copies/reaction) in qPCR assays.
- Demonstrated distinct and reproducible allelic discrimination with homogeneity coefficients of variation below 5%.
- Confirmed plasmid stability across generations and storage, with consistent SNP detection across various platforms and assays.
Conclusions:
- Developed plasmid-based reference materials show excellent analytical performance, stability, and reproducibility for CYP2D6 SNP detection.
- Cross-platform compatibility of these materials facilitates standardization of pharmacogenomic assays.
- Validated reference materials provide a cost-effective, accurate standard for pharmacogenomic testing, enhancing assay precision and clinical utility.
