New allele-specific real-time PCR system for warfarin dose genotyping equipped with an automatic interpretative function that allows rapid, accurate, and user-friendly reporting in clinical laboratories

Abstract

Introduction: The optimal dose of the oral anticoagulant warfarin varies with polymorphisms of the vitamin K epoxide reductase complex subunit 1 (VKORC1) and cytochrome P450 2C9 (CYP2C9) genes. A fast and reliable method of warfarin dose adjustment is required to prevent serious hemorrhagic or thrombotic complications. The aim of this study is to develop and validate a new warfarin dose genotyping system with an automatic interpretation function.

Materials and methods: Four VKORC1 and two CYP2C9 SNPs were genotyped by real-time PCR using allele-specific primers and probes. Multiple reactions that included internal positive controls were performed in each well, and an automatic interpretative algorithm was developed. This system was validated using 82 clinical specimens previously genotyped by PCR-direct sequencing. The analytical time of the method was calculated.

Results: No interference was observed when multiple samples were included in each reaction, with all internal positive control reactions being successful. In the genotyping algorithm, Ct differences <2 and ≥2 identified heterozygotes and homozygotes, respectively. All results obtained were concordant with those of the reference method. The overall analytical time for assay of 12 specimens was around 3 hours.

Conclusion: This rapid, accurate, and user-friendly genotyping system improves the efficacy and safety of anticoagulation therapy in clinical practice.