Development and Analytical Validation of a Laboratory‐Customized
TaqMan
‐
MGB
Probe Method for
CY
Yaqun Liu, Lianghui Chen, Xuanyi Zheng, Peikui Yang, Yicun Chen, Chengsong Xie, Zhenxia Zhang, Yuzhong Zheng ABSTRACT
Background
Genetic polymorphisms in CYP2C19 influence the metabolism of multiple clinically important drugs. Although TaqMan‐based genotyping of CYP2C19 *2 and CYP2C19 *17 is well established, open and locally implementable workflows remain useful for laboratory validation and application. This study aimed to develop and analytically validate a laboratory‐customized, open‐sequence TaqMan‐MGB assay for detecting CYP2C19 *2 (rs4244285, NM_000769.4:c.681G > A) and CYP2C19 *17 (rs12248560, NM_000769.4:c.‐806C > T).
Methods
Specific primers and allele‐specific TaqMan‐MGB probes were designed. The assay was evaluated for primer/probe performance, specificity, analytical sensitivity, candidate limit of detection (LOD), and repeatability. Preliminary clinical concordance was assessed using 20 dried blood spot (DBS) samples, with Sanger sequencing as the reference.
Results
The assay discriminated wild‐type, heterozygous, and homozygous mutant genotypes for CYP2C19 *2 and wild‐type and heterozygous genotypes for CYP2C19 *17 in tested clinical samples. Using plasmid templates, candidate LODs were 1.17 × 10 2 copies/μL for CYP2C19 *2 and 0.94 × 10 3 copies/μL for CYP2C19 *17. Repeatability testing with plasmid templates and representative DBS‐derived clinical genomic DNA showed coefficients of variation below 10%. All 20 DBS samples were concordant with Sanger sequencing, corresponding to 100% observed sample‐level concordance and an exact 95% confidence interval of 83.2%–100.0%.
Conclusion
The customized TaqMan‐MGB assay showed acceptable preliminary analytical specificity, sensitivity, and repeatability. Its value lies in the laboratory‐customized, open‐sequence design and DBS‐compatible workflow. Because the clinical set was limited and lacked the CYP2C19 *17 TT genotype, the findings should be regarded as preliminary. Larger studies including all relevant genotypes, additional CYP2C19 alleles, and diverse populations are needed before routine clinical implementation or comprehensive CYP2C19 phenotype assignment.