Abstract:

Objective To establish Taqman-MGB real-time fluorescence polymerase chain reaction (PCR) for determining CYP3A5 rs776746 site single nucleotide polymorphism (SNP), and to analyze the influence on the metabolism of tacrolimus. Methods The primers and double-labeled probes were designed according to the genotype of CYP3A5 rs776746 site, and the Taqman-MGB real-time fluorescence PCR for SNP was established and evaluated. A total of 170 whole blood DNA samples from patients who took tacrolimus were collected, and CYP3A5 genotype was determined by Taqman-MGB real-time fluorescence PCR. The relationship between CYP3A5 genotype and tacrolimus concentration to dose ratio was analyzed. Results The established Taqman-MGB method can discriminate CYP3A5 rs776746 site efficiently with good reproducibility and specificity and had a detection limit of 0.01 ng for DNA. The results of Taqman-MGB method were consistent with those of sequencing. Among the 170 samples, 18 samples belonged to CYP3A5*1/*1(10.59%), 65 samples were CYP3A5*1/*3(38.24%) and 87 samples were CYP3A5*3/*3(51.18%). Among the samples collected, 104 samples belonged to renal allograft recipients, and the mean tacrolimus concentrations to dose ratios for 3 genotypes were (52.47±35.96), (77.79±33.80) and (134.80±79.35) (ng/mL)/(mg·kg^-1·d^-1), and there was statistical significance (P<0.05). Conclusions The Taqman-MGB method for determining CYP3A5 rs776746 site SNP is established, which has high sensitivity, specificity and reproducibility and is a rapid method for clinical practice. Through primary analysis, the existence of CYP3A5*3 genotype do weaken the ability of metabolizing tacrolimus.

Key words: Taqman-MGB real-time fluorescence polymerase chain reaction, Methodology evaluation, Cytochrome P450, CYP3A5, Single nucleotide polymorphism