基于焦磷酸测序的CYP2C19*2、CYP2C19*3基因分型方法性能验证

Performance verification of CYP2C19*2 and CYP2C19*3 genotyping method based on pyrophosphate sequencing

目的对细胞色素P4502C19(CYP2C19)*2和CYP2C19*3单核苷酸多态性分型的实验室自建焦磷酸测序法进行性能验证,确保其能为临床提供准确可靠的结果。方法通过对梯度稀释的杂合型基因组DNA(gDNA)标本(10.0、2.0、0.4 ng/μL)进行检测,评价方法的最低检测浓度;比较焦磷酸测序和Sanger测序结果,评价方法的准确度;将经焦磷酸测序验证的野生型gDNA标本进行Sanger测序,评价方法的特异度;对杂合型gDNA标本重复检测4次,评价方法的重复性。结果焦磷酸测序检测CYP2C19*2和CYP2C19*3单核苷酸多态性的最低检测浓度为0.4 ng/μL;20份临床gDNA标本的焦磷酸测序结果与Sanger测序结果一致,准确度为100%;10份野生型的临床gDNA标本经Sanger测序验证均为野生型,特异度为100%;分别对CYP2C19*2和CYP2C19*3的杂合型gDNA标本重复检测4次,重复性好,变异系数均<5%。结论焦磷酸测序检测CYP2C19*2和CYP2C19*3单核苷酸多态性的结果准确可靠,能够为临床使用氯吡格雷、质子泵抑制剂等药物提供个体化指导。

Objective The performance of the self-built pyrophosphate sequencing method for single nucleotide polymorphism typing of cytochrome P4502C19(CYP2C19)*2 and CYP2C19*3 was verified to ensure that it can provide accurate and reliable results for clinical use.Methods The lowest detection concentration of the method was evaluated by detecting the heterozygous genomic DNA(gDNA)samples(10.0,2.0,0.4 ng/μL)with gradient dilution.The results of pyrophosphate sequencing and Sanger sequencing were compared to evaluate the accuracy of the method.Carried out Sanger sequencing on the wild type gDNA samples verified by pyrophosphate sequencing to evaluate the specificity of the method.The heterozygous gDNA samples were detected 4 times to evaluate the repeatability of the method.Results The minimum detection concentration of single nucleotide polymorphisms of CYP2C19*2 and CYP2C19*3 detected by pyrophosphate sequencing was 0.4 ng/μL.The pyrophosphate sequencing results of 20 clinical gDNA samples were consistent with Sanger sequencing,with an accuracy of 100%.A total of 10 clinical gDNA samples of wild type were confirmed as wild type by Sanger sequencing,with specificity of 100%.The heterozygous gDNA samples of CYP2C19*2 and CYP2C19*3 were detected 4 times,with good repeatability and coefficient of variation less than 5%.Conclusion The results of pyrophosphate sequencing for the detection of CYP2C19*2 and CYP2C19*3 single nucleotide polymorphism are accurate and reliable,which could provide individualized guidance for the clinical use of clopidogrel,proton pump inhibitors and other drugs.