Genotyping Of ADRB1,CYP2D6,CYP3A5 And NPPA By Multi-primers Allelic Discrimination

Objective: To discriminate genotypes of ADRB1,CYP2D6,CYP3A5,and NPPA a multiplex allele-specific(AS)qPCR analysis is developed,which can guide the delivery of anti-hypertension drugs such as beta adrenoceptor blockers,calcium antagonists,and diuretics.Methods: To establish a multiplex amplification analysis for genotyping of ADRB1,CYP2D6,CYP3A5 and NPPA,studied content covered following:(a)Mismatched AS Fprimers,R-primers and fluorescent probes were designed;(b)In this study,genotype was determined by value of ΔCq(the difference in threshold cycle between the wild-type Fprimer amplification assay and the mutant-type F-primer amplification assay).Mismatch AS F-primers were screened to enable maximization of ΔCq by using uniplex qPCR analysis to detect plasmids and human genomic DNA;(c)Agreement analysis considering NGS and Sanger as a reference method was implemented to validate the multiplex AS qPCR analysis employing selected F-primers;(d)Finally,analytical performance comprising detection range,reproducibility,cross-reactivity,interfering and stability was evaluated.Results: F-primers selected to identify single nucleotide polymorphism(SNP)appeared well specific.No statistically difference was found between the multiplex AS qPCR,NGS and Sanger sequencing.The detection range of the multiplex qPCR analysis was from 1.25 ng to 40 ng.Coefficient of variation(CV)for inter-assay or intra-assay was ≤ 5%.No significant cross-reactivity was observed.Homologous genes and oral inclusions did not affect genotyping outcomes.The stability assessment also appeared well results.Conclusions: The multiplex qPCR analysis for discrimination of genotypes of ADRB1,CYP2D6,CYP3A5 and NPPA is simple,accurate and low-cost,which can effectively guide hypertensive patients to rationally take β-adrenoceptor blockers,calcium antagonists and diuretics and other related drugs to avoid side-effect.