尿液游离核酸的大小分布情况及检测方式选择  

The size distribution and analytical methods of urinary cell-free DNA

作　　者：张鑫 邹聪 刘南 钱开宇 肖宇 朱源 ZHANG Xin;ZOU Cong;LIU Nan;QIAN Kai-yu;XIAO Yu;ZHU Yuan(Department of Biological Repositories,Zhongnan Hospital of Wuhan University,Hubei 430071,China)

机构地区：[1]武汉大学中南医院生物样本库,湖北430071

出　　处：《中国医药生物技术》2023年第2期122-126,共5页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金面上项目(82273065)。

摘　　要：目的探究泌尿系疾病患者尿液样本中游离核酸(cfDNA)的质量以及不同原理检测方法的选择。方法采用试剂盒提取尿液上清中的cfDNA,使用Nanodrop One、Qubit 4、4200 TapeStation及PCR对样本进行检测,查看其纯度、浓度及片段大小。结果467例尿液cfDNA样本浓度的中位数为16.70 ng/μl,最大值1710 ng/μl。仅有50%的样本纯度较高,A260/A280值在1.70~2.00之间,对于纯度较高的样本,不同仪器测得的浓度值比较接近。尿液cfDNA片段大小主要是180 bp左右且经常可见约180 bp整数倍的梯度条带,大分子量核酸片段分布在1.5 kb及8 kb左右。以游离核酸为模板,可扩增出不同大小的β球蛋白基因片段。结论不同来源的尿液上清中cfDNA浓度差异较大,大分子量核酸片段出现的频率较多,尿液中盐类等杂质可能导致cfDNA样本纯度偏低。采用分光光度法测量尿液cfDNA浓度易出现偏差,而荧光计或生物分析仪可测得较准确浓度值。Objective To investigate the quality of urinary cell-free nucleic acid(cfDNA)from patients with urinary disease and select appropriate methods for cfDNA analysis.Methods The cfDNA was extracted from urine using a commercial kit based on magnetic beads and spin column.The purity,concentration and size of the cfDNA samples were assessed by Nanodrop One,Qubit 4,and Agilent 4200 TapeStation.The isolated cfDNA was amplified by polymerase chain reaction(PCR).Results Among 467 urinary cfDNA samples,the median concentration was 16.70 ng/μl and the maximum value was 1710 ng/μl.Only 50%samples had a 260/280 ratio value range 1.70 to 2.00,which is generally accepted as"pure"for DNA.The concentration values measured by different quantification methods were close when the cfDNA samples were pure.Most cfDNA fragments were about 180 base pairs or ladder-like bands displaying multiples of about 180 bp,and some were high-molecular weight in the form of about 1.5 kb or 8.0 kb.β-globulin gene fragments could be amplified when different cfDNA as templates.Conclusion The concentration of cfDNA in urine supernatant from different sources varies greatly,large molecular weight nucleic acid fragments occur more frequently,and impurities such as salts in urine may cause low purity of cfDNA samples.Spectrophotometric measurement of urine cfDNA concentrations is prone to deviation,while fluorometers or bioanalyzers can measure more accurate concentration values.

关 键 词：尿液游离核酸 生物样本库 质量控制 

分 类 号：R446.12[医药卫生—诊断学]