目的 建立1种简便易用的药物基因组学检测方法,并对其进行评价。方法 基于竞争性等位基因特异性聚合酶链式反应(KASP)技术,重新设计通用荧光探针,通过华法林个体化用药相关基因CYP2C9*3(rs1057910)的阳性序列和临床生物样本进行验证。结果 Cal Red 610、Quasar 670的KASP测定CYP2C9*3华法林个体化用药相关基因结果准确,每个反应中最低可检出31个拷贝,在小规模的初步验证中实现了100%的特异性和灵敏度。结论 Cal Red 610、Quasar 670 KASP在CYP2C9*3华法林个体化用药相关基因多态性检测中,具有高准确性、高通量、检测浓度范围宽等特点,满足生物样本的低成本临床检测的需求,比传统的Fam/Hex荧光更具优势,避免了进一步推广中对特定试剂的依赖,为进一步降低成本奠定了基础。
Abstract
OBJECTIVE To establish and evaluate a simple and easy-to-use method for pharmacogenomics. METHODS Based on the kompetitive allele-specific PCR(KASP) technology, the universal fluorescent probe was redesigned. The positive sequence of CYP2C9*3 (rs1057910) and clinical biological samples were used to verify the detection system. RESULTS The KASP of Cal Red 610 and Quasar 670 was accurate in the detection of CYP2C9*3 related genes of warfarin individualization, and the lowest 31 copies could be detected in each reaction, achieving 100% sensitivity and specificity in small-scale preliminary verification. CONCLUSION The KASP of Cal Red 610 and quasar 670 has the characteristics of high accuracy, high throughput and wide detection concentration range in the detection of CYP2C9*3 drug-related gene polymorphism, which meets the needs of low-cost clinical detection of biological samples, has more advantages than traditional Fam / Hex fluorescence, avoids the dependence on specific reagents in further promotion, and lays a foundation for further cost reduction set the foundation.
关键词
华法林 /
个体化用药 /
基因分型 /
竞争性等位基因特异性聚合酶链式反应
{{custom_keyword}} /
Key words
warfarin /
individualized drug use /
genotyping /
kompetitive allele-specific PCR(KASP)
{{custom_keyword}} /
中图分类号：
R915
{{custom_clc.code}}
({{custom_clc.text}})
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] ZHANG J H, PENG W X, DONG Q, et al. Individualized medication of warfarin by quantitative pharmacology[J]. Chin Pharm J (中国药学杂志), 2018, 53(19): 1703-1708.
[2] AOMORI T, YAMAMOTO K, OGUCHI-KATAYAMA A, et al. Rapid single-nucleotide polymorphism detection of cytochrome P450 (CYP2C9) and vitamin K epoxide reductase (VKORC1) genes for the warfarin dose adjustment by the SMart-amplification process version 2 [J]. Clin Chem, 2009, 55(4):804-812.
[3] WAN L Y, WEI J Y, DONG M R, et al. Correlation of CYP2C9 and VKORC1 polymorphisms with warfarin stable dose [J]. Chin Pharm (中国药师), 2019, 22(3):490-493.
[4] FENG P P, PENG W X, SHI X J, et al. Research progress on the effect of gene polymorphism on warfarin dose[J]. J China Pharm (中国药房), 2017, 28(11): 1581-1584.
[5] MATSUDA K. PCR-based detection methods for single-nucleotide polymorphism or mutation: real-time PCR and its substantial contribution toward technological refinement [J]. Adv Clin Chem, 2017, 80:45-72.
[6] KHANAL C, KULARATHNA M T, RAY J D, et al. Single nucleotide polymorphism analysis using KASP assay reveals genetic variability in rotylenchulus reniformis [J]. Plant Dis, 2019,103(8):1835-1842.
[7] SHITAYE G, GADISA E, GRIGNARD L, et al. Low and heterogeneous prevalence of glucose-6-phosphate dehydrogenase deficiency in different settings in Ethiopia using phenotyping and genotyping approaches [J]. Malar J, 2018, 17(1):281.
[8] ZHANG G, XU Q, LIU J, et al. Five P53 SNPs involved in low rectal cancer risk and prognosis in a Chinese population [J]. J Cancer, 2019, 10(7):1772-1780.
[9] HE C, HOLME J, ANTHONY J. SNP genotyping: the KASP assay [J]. Methods Mol Biol, 2014,1145:75-86.
[10] Fluorophore and BHQ [EB/OL]. [2020-08-20]. https://biosearchassets.blob.core.windows.net/assets/bti_bhq_selectionchart.pdf
[11] WU D Y, UGOZZOLI L, PAL B K, et al. Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia [J]. Proc Natl Acad Sci USA, 1989, 86(8):2757-2760.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
国家重大新药创制专项资助(2017ZX09304017);北京市医院管理局临床医学发展专项经费资助(ZYLX201805)
{{custom_fund}}
PDF(1277 KB)
