采用 EvaGreen 化学基于液滴数字 PCR 检测分析人血浆无细胞 DNA 片段大小分布。

Analysis of human blood plasma cell-free DNA fragment size distribution using EvaGreen chemistry based droplet digital PCR assays.

机构信息

Department of Obstetrics and Gynecology, University of Nebraska Medical Center, Omaha, NE, USA; Department of Research and Development, CFGenome, Omaha, NE, USA.

Department of Obstetrics and Gynecology, University of Nebraska Medical Center, Omaha, NE, USA.

出版信息

Clin Chim Acta. 2018 Aug;483:39-47. doi: 10.1016/j.cca.2018.04.017. Epub 2018 Apr 12.

DOI:10.1016/j.cca.2018.04.017

PMID:29655637

Abstract

BACKGROUND

Plasma cell-free DNA (cfDNA) fragment size distribution provides important information required for diagnostic assay development. We have developed and optimized droplet digital PCR (ddPCR) assays that quantify short and long DNA fragments. These assays were used to analyze plasma cfDNA fragment size distribution in human blood.

METHODS

Assays were designed to amplify 76,135, 490 and 905 base pair fragments of human β-actin gene. These assays were used for fragment size analysis of plasma cell-free, exosome and apoptotic body DNA obtained from normal and pregnant donors.

RESULTS

The relative percentages for 76, 135, 490 and 905 bp fragments from non-pregnant plasma and exosome DNA were 100%, 39%, 18%, 5.6% and 100%, 40%, 18%,3.3%, respectively. The relative percentages for pregnant plasma and exosome DNA were 100%, 34%, 14%, 23%, and 100%, 30%, 12%, 18%, respectively. The relative percentages for non-pregnant plasma pellet (obtained after 2nd centrifugation step) were 100%, 100%, 87% and 83%, respectively.

CONCLUSION

Non-pregnant Plasma cell-free and exosome DNA share a unique fragment distribution pattern which is different from pregnant donor plasma and exosome DNA fragment distribution indicating the effect of physiological status on cfDNA fragment size distribution. Fragment distribution pattern for plasma pellet that includes apoptotic bodies and nuclear DNA was greatly different from plasma cell-free and exosome DNA.

摘要

背景

血浆无细胞游离 DNA (cfDNA) 片段大小分布为诊断检测开发提供了重要信息。我们开发并优化了用于定量短片段和长片段 DNA 的液滴数字 PCR (ddPCR) 检测。这些检测用于分析人血液中的血浆 cfDNA 片段大小分布。

方法

设计了检测来扩增人β-肌动蛋白基因的 76,135、490 和 905 个碱基对片段。这些检测用于分析来自正常和妊娠供体的血浆无细胞游离、外泌体和凋亡小体 DNA 的片段大小。

结果

非妊娠血浆和外泌体 DNA 的 76、135、490 和 905 bp 片段的相对百分比分别为 100%、39%、18%、5.6%和 100%、40%、18%、3.3%。妊娠血浆和外泌体 DNA 的相对百分比分别为 100%、34%、14%、23%和 100%、30%、12%、18%。第二次离心后获得的非妊娠血浆沉淀的相对百分比分别为 100%、100%、87%和 83%。