双标记TaqMan探针实时荧光PCR法检测ALDH2基因型及初步临床应用
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摘要
目的评价双标记TaqMan探针实时荧光PCR法(dFQ-PCR)检测ALDH2基因型的可行性,并初步用于临床检测。方法对ALDH2基因型检测的dFQ-PCR进行方法学性能评价,包括准确度、检测下限、重复性和抗干扰能力,并在200例体检者中进行基因型检测。结果 ALDH2基因分型只需单管PCR扩增,操作简单,结果判断直观。与测序方法比较,准确性为100%,检测下限接近5 ng/μl,重复性好,常规浓度的干扰物均对基因型检测无干扰。本院200例体检者的GG、AG和AA基因型比例分别为60.0%、35.0%和4.5%,G和A等位基因频率分别为77.8%和22.2%,符合Hardy-Weinberg遗传平衡。结论 ALDH2基因型的dFQ-PCR检测方法单管扩增检测,具有操作简便快捷、结果判断简单、方法性能良好和所用仪器普及高等优点,适用于临床上大样本的ALDH2基因分型。
Objective To evaluate the feasibility of double-labeled TaqMan probes-based real-time PCR assay( dFQ-PCR) for detecting ALDH2 genotypes and apply it for clinical detection. Methods Methodological performances of dFQ-PCR for detecting ALDH2 genotypes,including the accuracy,limit of detection( LOD),repeatability and anti-jamming capability were evaluated. Meanwhile,the genotypes of 200 clinical samples from health examination were determined by dFQ-PCR.Results dFQ-PCR assay,which was a simple,rapid,and intuitive assay,can be performed in a single reaction to determine ALDH2 genotypes. Meanwhile,the accuracy was 100% compared with the sequencing method. The LOD reaches 5 ng/μl and the method also showed a high repeatability. Moreover,the genotyping results were not interfered with the conventional interfering substances in blood. The genotype frequencies of GG,AG and AA from 200 physical examination people in local area were 60. 0%,35. 0% and 4. 5%,respectively,and the G and A allele frequencies were 77. 8% and 22. 2%,respectively,which were in accordance with the genetic balance of Hardy-Weinberg. Conclusion The ALDH2 genotyping of dFQ-PCR in a single reaction has the advantages of easy operation,simple analysis,reliable performance and instrumental versatility,which is suitable for ALDH2 genotyping of batch clinical samples.
Objective To evaluate the feasibility of double-labeled TaqMan probes-based real-time PCR assay( dFQ-PCR) for detecting ALDH2 genotypes and apply it for clinical detection. Methods Methodological performances of dFQ-PCR for detecting ALDH2 genotypes,including the accuracy,limit of detection( LOD),repeatability and anti-jamming capability were evaluated. Meanwhile,the genotypes of 200 clinical samples from health examination were determined by dFQ-PCR.Results dFQ-PCR assay,which was a simple,rapid,and intuitive assay,can be performed in a single reaction to determine ALDH2 genotypes. Meanwhile,the accuracy was 100% compared with the sequencing method. The LOD reaches 5 ng/μl and the method also showed a high repeatability. Moreover,the genotyping results were not interfered with the conventional interfering substances in blood. The genotype frequencies of GG,AG and AA from 200 physical examination people in local area were 60. 0%,35. 0% and 4. 5%,respectively,and the G and A allele frequencies were 77. 8% and 22. 2%,respectively,which were in accordance with the genetic balance of Hardy-Weinberg. Conclusion The ALDH2 genotyping of dFQ-PCR in a single reaction has the advantages of easy operation,simple analysis,reliable performance and instrumental versatility,which is suitable for ALDH2 genotyping of batch clinical samples.
引文
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[3]中华人民共和国国家卫生和计划生育委员会.药物代谢酶和药物作用靶点基因检测技术指南(试行)概要[J].实用器官移植电子杂志,2015,3(5):257-267.
[4]刘维薇,关明.2013版《医学实验室质量和能力认可准则在基因扩增检验领域的应用说明》改版解读[J].中华临床实验室管理电子杂志,2013,1(1):36-40.
[5]Mizuno Y,Hokimoto S,Harada E,et al.Variant aldehyde dehydrogenase 2(ALDH2*2)is a risk factor for coronary spasm and ST-segment elevation myocardial infarction[J].J Am Heart Assoc,2016,5(5):e003247.
[6]Wang Y,Du F,Zhao H,et al.Synergistic association between two alcohol metabolism relevant genes and coronary artery disease among Chinese hypertensive patients[J].PLo S ONE,2014,9(7):e103161.
[7]Sung YF,Lu CC,Lee JT,et al.Homozygous ALDH2*2 is an independent risk factor for ischemic stroke in taiwanese men[J].Stroke,2016,47(9):2174-2179.
[8]Mizuno Y,Hokimoto S,Harada E,et al.Variant aldehyde dehydrogenase 2(ALDH2*2)in east asians interactively exacerbates tobacco smoking risk for coronary spasm-possible role of reactive aldehydes[J].Circ J,2016,81(1):96-102.
[9]Oniki K,Morita K,Watanabe T,et al.The longitudinal effect of the aldehyde dehydrogenase 2*2 allele on the risk for nonalcoholic fatty liver disease[J].Nutr Diabetes,2016,6:e210.
[10]Chang JS,Hsiao JR,Chen CH.ALDH2 polymorphism and alcoholrelated cancers in Asians:a public health perspective[J].J Biomed Sci,2017,24(1):19.
[11]Li R,Zhao Z,Sun M,et al.ALDH2 gene polymorphism in different types of cancers and its clinical significance[J].Life Sci,2016,147:59-66.
[12]Liu K,Song G,Zhu X,et al.Association between ALDH2Glu487Lys polymorphism and the risk of esophageal cancer[J].Medicine(Baltimore),2017,96(16):e6111.
[13]Mc Allister SL,Sun K,Gross ER.Developing precision medicine for people of East Asian descent[J].J Biomed Sci,2016,23(1):80.
[14]罗招凡,何嘉辉,方伟祯,等.广州地区心血管病患者CYP2C19*1、CYP2C19*17基因多态性对氯吡格雷疗效的影响[J].中国卫生检验杂志,2017,27(13):1831-1833,1837.
[15]孙宇晶,线海鹏,刘向祎,等.等位基因特异荧光PCR检测ALDH2基因多态性[J].检验医学,2017,32(4):267-271.
[16]徐娟,郑绯,姜楠,等.特异荧光PCR对人ALDH2基因型的研究[J].中国卫生检验杂志,2014,24(16):2391-2392,2395.
[17]Muramatsu T,Wang ZC,Fang YR,et al.Alcohol and aldehyde dehydrogenase genotypes and drinking behavior of Chinese living in Shanghai[J].Hum Genet,1995,96(2):151-154.