Screening, Confirmation and Validation of Mismatch AS F-Primers for Hypertension-Related Genotyping

In this study, screening, confirmation and validation of mismatch allele-specific (AS) forward (F)-primers are executed to establish a quadruplex amplification analysis (real-time PCR) for discrimination of CYP2D6*10, ADRB1, NPPA and CYP3A5*3 genotypes associated with hypertensive pharmacogenomics. To significantly distinguish heterozygote and homozygote, ΔCq (differences in threshold cycles between the wild-type F-primer amplification assay and the mutant-type F-primer amplification assay) was utilized to determine outcomes. Detection of plasmid by uniplex real-time PCR was used to screen the mismatch AS F-primers. Robustness assessment and agreement analysis were employed to confirm and validate initially selected F-primers, respectively. Robustness assessment confirmed that except of ADRB1 (0.7-0.9), amplification efficiency ranged from 0.9 to 1.1. No statistically significant difference was found between the analysis and NGS. Therefore, the optimized F-primer as polymorphism recognition molecules can benefit the genotyping guiding drug delivery in anti-hypertension treatment.