人卵泡液游离DNA提取及定量检测方法的比较  

Comparison of Extraction and Quantitative Method of Free DNA in Human Follicular Fluid

作　　者：池梅 刘宇[2,4] 申秋子 邹敏 李自立 张衷源 杜欣 张玲[2,3] CHI Mei;LIU Yu;SHEN Qiuzi(Obstetrics and Gynecology,Medical College of Wuhan University of Science and Technology,Wuhan Hubei 430062,China;Institute of Reproductive Health,Tongji Medical College,Huazhong University of Science and Technology,Wuhan Hubei 430000,China;Department of Gynecology,Hubei Maternal and Child Health Hospital,Wuhan Hubei 430070,China)

机构地区：[1]武汉科技大学医学院妇产科,湖北武汉430062 [2]华中科技大学同济医学院生殖健康研究所,湖北武汉430000 [3]华中科技大学同济医学院生殖医学中心,湖北武汉430000 [4]湖北省妇幼保健院妇科,湖北武汉430070

出　　处：《实用妇产科杂志》2023年第5期375-380,共6页Journal of Practical Obstetrics and Gynecology

基　　金：湖北省卫生健康委员会面上项目(编号:WJ2019M112)。

摘　　要：目的:比较不同的DNA提取方法、实时荧光定量PCR(qPCR)的引物及荧光染料对人卵泡液游离核DNA(cf-nDNA)和游离线粒体DNA(cf-mtDNA)定量检测的影响。方法:收集2018年3月~10月在华中科技大学同济医学院生殖医学中心进行体外受精/卵胞浆内单精子显微注射技术(IVF/ICSI)的40例患者卵泡液样本,分别用4种不同的方法(方法一:BeaverBeads^(TM) Circulating DNA试剂盒、方法二:蛋白酶K(PK)法、方法三:Hieff^(■) qPCR SYBR^(■) Green Master Mix和方法四:KOD SYBR^(■) qPCR Mix)提取和纯化卵泡液中的游离DNA(cf-DNA)样本;用4种cf-nDNA引物(ALU115、B2MF1、GAPDH和β-globin)和4种cf-mtDNA引物(ND1-primer1、ND1-primer2、hmito3和hmito5)分别定量检测卵泡液cf-nDNA和cf-mtDNA水平;比较qPCR实验中SYBR Green化学染料和TaqMan探针两种方法在定量检测上的差异。结果:提取cf-nDNA的浓度按以下顺序排列:方法一>方法四>方法二>方法三(P<0.05),提取cf-mtDNA的浓度按以下顺序排列:方法一>方法四>方法三>方法二(P<0.05);样本中cf-nDNA的扩增浓度按照以下顺序排列:ALU115>GAPDH>β-globin>B2MF1(P<0.05),4种cf-nDNA引物无相关性(P>0.05)。cf-mtDNA的扩增浓度按以下顺序排列:ND1-primer1>ND1-primer2>hmito5>hmito3(P<0.05)。ND1-primer1和ND1-primer2有很强的相关性(r=0.517,P<0.05),hmito3和hmito5也有很强的相关性(r=0.989,P<0.05);在以β-globin和ND1-primer1为引物的qPCR实验中,TaqMan探针法检测的Ct值高于SYBR Green化学染料法(P<0.05)。结论:人卵泡液样本的提取方法、引物和荧光染料的选择均影响cf-nDNA和cf-mtDNA的定量,建议根据不同的研究目的选择合适的方法。Objective:To compare the effects of different DNA extraction methods,primers and fluorescent dyes for real-time quantitative PCR(qPCR)on the quantitative detection of free nuclear DNA(cf-nDNA)and free mitochondrial DNA(cf-mtDNA)in human follicular fluid.Methods:Follicular fluid samples from 40 patients who un-derwent in vitro fertilization/intracytoplasmic single sperm microinjection(IVF/ICSI)at the Center for Reproductive Medicine,Tongji Medical College,Huazhong University of Science and Technology from March 2018 to October 2018 were collected,using four different methods(Method one:BeaverBeads^(TM) Circulating DNA kit,Method two:Proteinase K(PK)method,Method three:Hieff^(■) qPCR SYBR^(■) Green Master Mix and Method four:KOD SYBR^(■) qPCR Mix)to extract and purify free DNA(cf-DNA)samples from follicular fluid.Four cf-nDNA primers(ALU115,B2MF1,GAPDH andβ-globin)and four cf-mtDNA primers(ND1-primer1,ND1-primer2,hmito3 and hmito5)were used to quantify follicular fluid cf-nDNA and cf-mtDNA levels,respectively.the SYBR Green chemical dye and TaqMan probes were used to compare the differences in quantification in qPCR assays.Results:The concentra-tions of extracted cf-nDNA were in the following order:Method one>Method four>Method two>Method three(P<0.05).The concentrations of extracted cf-mtDNA were in the following order:Method one>Method four>Method three>Method two(P<0.05).The amplification concentrations of cf-nDNA in the samples was in the fol-lowing order,ALU115>GAPDH>β-globin>B2MF1(P<0.05),and the four cf-nDNA primers were not correlated(P>0.05).The amplification concentrations of cf-mtDNA was in the following order,ND1-primer1>ND1-primer2>hmito5>hmito3(P<0.05).ND1-primer1 and ND1-primer2 had a strong correlation(r=0.517,P<0.05),and hmito3 and hmito5 also had a strong correlation(r=0.989,P<0.05).In qPCR assays usingβ-globin and ND1-primer1 as primers,the Ct value detected by TaqMan probe method was higher than that detected by SYBR Green chemical dye method(P<0.05).Conclusions:Different DNA extraction

关 键 词：游离核DNA 游离线粒体DNA 人卵泡液 检测方法 

分 类 号：R711[医药卫生—妇产科学]