CYP3A5 rs776746 Genotyping by SYBR Green-Based Real-Time Multiplex Polymerase Chain Reaction Combined with Melting Curve Analysis Using Tm-Shift Method

Background: Cytochrome P450 3A subfamily members (CYP3A5) affects drug reactions and susceptibility to diseases.The homozygotic variant rs776746 (CYP3A5 *3/*3; NM_000777.5:c.219-237A>G)genotype is a nonexpressor of CYP3A5 and metabolizes CYP3A5 substrates more slowly than CYP3A5expressor genotypes (CYP3A5*1/*1 and *1/*3).A rapid and reliable CYP3A5 genotyping is necessary to reduce the risk of adverse drug reactions.Methods: A total of 101 randomly collected blood samples from hospitalized patients between May 2022 and December 2022 were used to evaluate this rapid CYP3A5 genotyping technique.One allele-nonspecific forward primer and two allele-specific reverse primers (a rs776746 G allele-specific primer with GC-rich sequences and a rs776746 A allele-specific primer without GCrich sequences) were used for CYP3A5 genotyping.We performed Sanger sequencing to confirm the results of real-time multiplex allele-specific polymerase chain reaction (PCR) and melting curve analysis.Results: The duration of the analysis of the CYP3A5 genotyping, except DNA extraction, was approximately 1.5 hours.The mean melting temperatures were 79.1℃ and 75.1℃, which were characteristic of rs776746 G allelespecific amplicon and rs776746 A allele-specific amplicon, respectively.Frequencies of CYP3A5*1/*1, CYP3A5*1/*3, and CYP3A5*3/*3 were 7 (6.9%),40 (39.6%), and 54 (53.5%) in 101 samples, respectively.The concordance rate between the real-time PCR combined with melting curve analysis and Sanger sequencing was 100%.Conclusions: CYP3A5 genotyping by SYBR Green-based real-time multiplex PCR combined with melting curve analysis using three unlabeled primers including the melting temperature-shift primer in a single tube is a rapid and reliable technique for routine laboratory testing.