LNA-taqman探针实时荧光PCR快速检测CYP4F2-C1347T,CYP2C9*3,VKORC1-C1173T与VKORC1-G1639A基因多态性方法的建立  

Establishment of LNA-taqman Quantitative Real-time PCR for Detecting CYP4F2-C1347T,CYP2C9*3,VKORC1-C1173T and VKORC1-G1639A Genes Polymorphism

作　　者：尹栩芳 蒋卫霞 饶品彬 

机构地区：[1]上海交通大学医学院附属苏州九龙医院检验科,江苏苏州215000 [2]苏州吉玛基因股份有限公司,江苏苏州215000

出　　处：《现代检验医学杂志》2017年第6期1-5,10,共6页Journal of Modern Laboratory Medicine

摘　　要：目的建立一种基于LNA-taqman探针实时荧光PCR检测华法林剂量相关基因型维生素K单氧酶CYP4F2,维生素K环氧化物还原酶复合体亚单位1(VKORC1)和细胞色素P4502C9(CYP2C9)的方法。方法针对CYP4F2-1347C>T,CYP2C9*3,VKORC1-1173C>T与VKORC1-1639G>A四个单核苷酸多态性(SNP)位点,分别设计一套等位基因特异性探针与引物,并对LNA-taqman探针特异性进行评价。提取150例接受华法林治疗患者外周血样本基因组DNA进行检测,同时对部分实时荧光PCR产物样品进行验证。结果 (1)LNA-taqman探针检测方法特异性高,没有交叉性。(2)150例患者中,CYP4F2-1347C>T基因型CC,CT和TT分别有87,56和7例,各占58%,37.3%和4.7%;CYP2C9*3基因型*1/*1和*1/*3分别有142和8例,各占94.7%和5.3%,未检出纯合子*3/*3基因型;VKORC1-1173C>T基因型TT,TC和CC分别有127,20和3例,各占84.7%,13.3%和2%;VKORC1-1639G>A基因型AA,AG和GG分别有124,23和3例,各占82.7%,15.3%和2%。结论基于LNA-taqman探针的实时荧光PCR分型方法具有操作简便,价格低廉,结果准确可靠等特点,适于临床实验室对CYP4F2,CYP2C9*3,VKORC1的基因分型。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-l173C〉T and VKORCl-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. O Among the 150 patients, the cases of CC, CT and TT genotypeof 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%0） ,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-Cl173T and VKORC1-G1639A genotyping in a clinic laboratory.

关 键 词：华法林 CYP4F2 CYP2C9 VKORCl 

分 类 号：Q503[生物学—生物化学]