ABSTRACT
Objective To establish a LNA-taqman-based real-time PCR assay for detecting gene polymorphisms of Vitamin K monooxygenase CYP4F2,vitamin K epoxide reductase complex subunit 1(VKORC1)and cytochrome P450 2C9(CYP2C9), associated with Warfarin optimal dosage.Methods A set of allele-specific PCR primers and probes was designed for each single nucleotide polymorphism(SNP)of CYP4F2-1347C>T,CYP2C9*3,VKORC1-1173C>T and VKORC1-1639G>A, and the specificity of LNA-taqman probe PCR was evaluated.Genomic DNA of peripheral blood samples from 150 patients with treated with warfarin was extracted,and the some PCR products were verified with sequencing.Results ①LNA-taq-man-based real-time PCR assay was highly specificity,no overla.②Among the 150 patients,the cases of CC,CT and TT genotype of CYP4F2-1347C>T were 87(58%),56(37.3%)and 7(4.7%).The cases of *1/*1 and *1/*3 genotype of CYP2C9*3 were 142(94.7%)and 8(5.3%),*3/*3 genotype was not detected.The cases of TT,TC and CC genotype of VKORC1 1173C>T were 127(84.7%),20(13.3%)and 3(2%).The cases of AA,AG and GG genotype of VKORC1 1639G>A were 124(82.7%),23(15.3%)and 3(2%),respectively.Conclusion The LNA-taqman-based real-time PCR as-say is convenient,inexpensive,accurate and will be useful for CYP4F2-C1347T,CYP2C9* 3,VKORC1-C1173T and VKORC1-G1639A genotyping in a clinic laboratory.