基于级联核酸侵入反应的real-time PCR法检测咽拭子样本中UGT1A1*6基因多态性

doi:10.16288/j.yczz.18-074

遗传 ›› 2018, Vol. 40 ›› Issue (8): 668-675.doi: 10.16288/j.yczz.18-074

基于级联核酸侵入反应的real-time PCR法检测咽拭子样本中UGT1A16*基因多态性

张婕妤,初亚男,邹秉杰,张晏洁,封利颖()

南京总医院药理科,南京 210002

收稿日期:2018-03-26 修回日期:2018-05-17 出版日期:2018-08-16 发布日期:2018-05-31
通讯作者: 封利颖 E-mail:flying870831@163.com
作者简介:张婕妤,硕士研究生,药师,研究方向：药物基因组学。E-mail: zjy881021@126.com
基金资助:
南京总医院院管课题(2015043);南京总医院院管课题(2015042);国家自然科学基金项目资助(81703474)

Detection of UGT1A16* single nucleotide polymorphism in oral swap samples using the cascade invader assay-based real-time PCR

Zhang Jieyu,Chu Yanan,Zou Bingjie,Zhang Yanjie,Feng Liying()

Department of Pharmacology, Nanjing General Hospital, Nanjing 210002, China

Received:2018-03-26 Revised:2018-05-17 Online:2018-08-16 Published:2018-05-31
Contact: Feng Liying E-mail:flying870831@163.com
Supported by:
Supported by Nanjing General Hospital Fund(2015043);Supported by Nanjing General Hospital Fund(2015042);National Natural Science Foundation of China(81703474)

摘要/Abstract

摘要：

伊立替康(irinotecan)不良反应与UGT1A1*6单核苷酸多态性相关,目前应用的单核苷酸多态性检测方法具有耗时长、开盖易污染、操作繁琐等缺点,因此需建立一种操作简便且不易污染的适合临床应用的新方法。本研究利用3种已知基因型的DNA样本建立基于级联核酸侵入反应的real-time PCR法检测UGT1A1*6多态性的最佳反应体系,并对其检测口腔咽拭子样本的灵敏度和准确性进行了考察。结果显示,本文成功建立了基于级联核酸侵入反应的UGT1A1*6基因多态性检测方法,可用于咽拭子样本的分型检测,其灵敏度达到6 ng DNA,分型准确性达到100%。因其取样方便对人体无创,单管闭管检测不易产生交叉污染,具有用于临床检测伊立替康的个体化用药相关UGT1A1*6基因多态性的潜力。

关键词: 核酸侵入反应, UGT1A1*6, 基因多态性, 伊立替康

Abstract:

The adverse reaction to irinotecan is related to the single nucleotide polymorphism (SNP) of UGT1A1*6 genotype. The current SNP detection methods have various disadvantages, including time-consuming procedures, high- risk cross-contamination, and cumbersome operation. Hence, it is necessary to establish a new method suitable for clinical application, which is easy and simple to detect SNP with minimal risk for cross-contamination. In this study, a cascade invader assay-based real-time PCR, for UGT1A1*6 genotyping has been established by optimizing reaction conditions with DNA samples of three genotypes. The sensitivity and accuracy of the method were evaluated with DNAs derived from oral swab samples. The results showed that the method could detect the UGT1A1*6 genotypes from the oral swab samples with a detection limit of 6 ng genomic DNA with 100% accuracy. Due to its convenient and non-invasive sampling, single close-tube operation, and minimal risk for cross-contamination, the method has the potential in clinical application for individualized detection of drug-related UGT1A1*6 polymorphism and reaction to irinotecan.

Key words: invader assay, UGT1A1*6, polymorphism, irinotecan

张婕妤,初亚男,邹秉杰,张晏洁,封利颖. 基于级联核酸侵入反应的real-time PCR法检测咽拭子样本中UGT1A1*6基因多态性[J]. 遗传, 2018, 40(8): 668-675.

Zhang Jieyu,Chu Yanan,Zou Bingjie,Zhang Yanjie,Feng Liying. Detection of UGT1A1*6 single nucleotide polymorphism in oral swap samples using the cascade invader assay-based real-time PCR[J]. Hereditas(Beijing), 2018, 40(8): 668-675.

参考文献

[1]	Federico I, Samir DU, Lalitha I, Chen PX, Soma D, Masha K, Theodore K, Linda J, Jacqueline R, Charles MR, Everett EV, Mark JR . Genetic variants in the UDP-glucuronosyl transferase 1A1 gene predict the risk of severe neutropenia of irinotecan. J Clin Oncol, 2004,22(8):1382-1388.
[2]	Cheng L, Li M, Hu J, Ren W, Xie L, Sun ZP, Liu BR, Xu GX, Dong XL, Qian XP . UGT1A1*6 polymorphisms are correlated with irinotecan-induced toxicity: A system review and meta-analysis in Asians. Cancer Chemother Pharmacol, 2014,73(3):551-560.
[3]	Naminatsu T, Yousuke N, Hiroyuki I, Takashi S, Yumiko S, Daiya T, Tsuyoshi H, Rie U, Suguru M, Koji M, Dai M, Kazumichi K, Hirofumi K, Natsuyo Y, Naoki S, Kenji H, Hideaki I, Minoru T, Yutaka Y, Kazuhiko K . Uridine diphosphate glucuronosyl transferase 1 family polypeptide A1 gene (UGT1A1) polymorphisms are associated with toxicity and efficacy in irinotecan monotherapy for refractory pancreatic cancer. Cancer Chemother Pharmacol, 2013,71(1):85-92.
[4]	Nie YC, Zhang C, Liu YN, Huang JP, Jiao HT, Wu D, Zhou HG . AS-PCR assay for 20 mtDNA SNP typing and haplotype frequency. Journal of Forensic Medicine, 2014,30(2):96-100, 109.
[4]	聂燕钗, 张晨, 刘亚楠, 黄江平, 焦海涛, 吴丹, 周怀谷 . AS-PCR技术检测20个mtDNA SNP位点及单倍型频率. 法医学杂志, 2014,30(2):96-100, 109.
[5]	Olsson LM, Johansson ÅC, Gullstrand B, Jönsen A, Saevarsdottir S, Rönnblom L, Leonard D, Wetterö J, Sjöwall C, Svenungsson E, Gunnarsson I, Bengtsson AA , Holmdahl R1. A single nucleotide polymorphism in the NCF1 gene leading to reduced oxidative burst is associated with systemic lupus erythematosus. Ann Rheum Dis, 2017,76(9):1607-1613.
[6]	Tayel SI, Fouda EAM, Elshayeb EI, Eldakamawy ARA, El-Kousy SM . Biochemical and molecular study on interleukin-1β gene expression and relation of single nucleotide polymorphism in promoter region with type 2 diabetes mellitus. J Cell Biochem, 2018, doi: 10.1002/jcb. 26667.[Epub ahead of print]
[7]	Hwang MT, Landon PB, Lee J, Choi D, Mo AH, Glinsky G, Lal R . Highly specific SNP detection using 2D graphene electronics and DNA strand displacement. Proc Natl Acad Sci USA, 2016,113(26):7088-7093.
[8]	Dhas DB, Ashmi AH, Bhat BV, Parija SC, Banupriya N . Modified low cost SNP genotyping technique using cycle threshold (Ct) & melting temperature (Tm) values in allele specific real-time PCR. Indian J Med Res, 2015,142(5):555-562.
[9]	Zheng ML, Qi XM, Tong H, Liu YL, Zou BJ, Song QX, Zhou GH . Detection of single nucleotide polymorphism by real-time polymerase chain reaction coupled with high specific invader assay in single tube. Chin J Anal Chem, 2015,43(7):1001-1008.
[9]	郑梦琳, 齐谢敏, 童欢, 刘云龙, 邹秉杰, 宋沁馨, 周国华 . 实时荧光聚合酶链式反应偶联高特异性核酸侵入反应检测单核苷酸多态性. 分析化学, 2015,43(7):1001-1008.
[10]	Nakahara H, Sekiguchi K, Hosono N, Kubo M, Takahashi A, Nakamura Y, Kasai K . Criterion values for multiplex SNP genotyping by the invader assay. Forensic Sci Int Genet, 2010,4(2):130-136.
[11]	Liu YL, Zou BJ, Wu HP, Chu YN, Zhou GH . Specificity improvement of invader assay by introducing an artificially mismatched base into the probe. Anal Methods, 2015,7(23):9779-9784.
[12]	Fuchs CS, Moore MR, Harker G, Villa L, Rinaldi D, Hecht JR . Phase III comparison of two irinotecan dosing regimens in second-line therapy of metastatic colorectal cancer. J Clin Oncol, 2003,21(5):807-814.
[13]	Li M, Wang Z, Guo J, Liu J, Li C, Liu L, Shi H, Liu L, Li H, Xie C, Zhang X, Sun W, Fang S, Bi X . Clinical significance of UGT1A1 gene polymorphisms on irinotecan- based regimens as the treatment in metastatic colorectal cancer. Onco Targets Ther, 2014,7:1653-1661.
[14]	Liu XQ, Zhu SH, Zou BJ, Ma YJ, Zhou GH . Development of 3-enzyme pyrosequencing system and its application in rapid diagnosis of Down’s syndrome. Hereditas (Beijing), 2010,32(5):517-523.
[14]	刘夕群, 朱术会, 邹秉杰, 马寅姣, 周国华 . 三酶焦测序体系的建立及其在唐氏综合征快速诊断中的应用. 遗传, 2010,32(5):517-523.
[15]	Cao CG, Sun HY, Zhou FF, Wang SM, Chen HY, Lu DR . Duplex genotyping of CYP2C192 and CYP2C193 by high-resolution melting curve analysis. Hereditas (Beijing), 2013,35(7):923-930.
[15]	曹春鸽, 孙海燕, 周芳芳, 王诗铭, 陈红岩, 卢大儒 . 应用HRM技术对CYP2C192和CYP2C193进行双重SNP分型. 遗传, 2013,35(7):923-930.

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基于级联核酸侵入反应的real-time PCR法检测咽拭子样本中UGT1A16*基因多态性

Detection of UGT1A16* single nucleotide polymorphism in oral swap samples using the cascade invader assay-based real-time PCR

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基于级联核酸侵入反应的real-time PCR法检测咽拭子样本中UGT1A1*6基因多态性

Detection of UGT1A1*6 single nucleotide polymorphism in oral swap samples using the cascade invader assay-based real-time PCR

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基于级联核酸侵入反应的real-time PCR法检测咽拭子样本中UGT1A16*基因多态性

Detection of UGT1A16* single nucleotide polymorphism in oral swap samples using the cascade invader assay-based real-time PCR