新型血浆外参照设计及核酸检测分析的研究

古骄阳; 牛凯; 余永新; 郑林鹏; 杨峤; 张露萍; 孙建国

doi:10.16016/j.1000-5404.201812172

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新型血浆外参照设计及核酸检测分析的研究

基础医学 | 更新时间：2026-01-09

- 新型血浆外参照设计及核酸检测分析的研究
- Design of a new plasma external reference and analysis of nucleic acid detection
- 第三军医大学学报 2019年41卷第13期页码：1239-1245
- 作者机构：
  
  400037 重庆，陆军军医大学(第三军医大学)第二附属医院全军肿瘤研究所
- 作者简介：
  
  [孙建国，E-mail：sunjg09@aliyun.comSUN Jianguo, E-mail: sunjg09@aliyun.com
- 基金信息：
  
  国家自然科学基金青年科学基金(81602688);重庆市自然科学基金(CSTC2016jcyjA2013)
- DOI：10.16016/j.1000-5404.201812172
  中图分类号： R394-33;R394.3;R446.11
- 收稿：2018-12-21，
  
  修回：2019-03-14，
  
  纸质出版：2019-07-15
- 稿件说明：
移动端阅览
古骄阳, 牛凯, 余永新, 等. 新型血浆外参照设计及核酸检测分析的研究[J]. 第三军医大学学报, 2019,41(13):1239-1245.

Jiaoyang GU, Kai NIU, Yongxin YU, et al. Design of a new plasma external reference and analysis of nucleic acid detection[J]. Journal of Third Military Medical University, 2019, 41(13): 1239-1245.
古骄阳, 牛凯, 余永新, 等. 新型血浆外参照设计及核酸检测分析的研究[J]. 第三军医大学学报, 2019,41(13):1239-1245. DOI： 10.16016/j.1000-5404.201812172.

Jiaoyang GU, Kai NIU, Yongxin YU, et al. Design of a new plasma external reference and analysis of nucleic acid detection[J]. Journal of Third Military Medical University, 2019, 41(13): 1239-1245. DOI： 10.16016/j.1000-5404.201812172.

摘要

目的

设计新型血浆外参照(plasma external reference，PLACON)，用于人类血浆mRNA、miRNA的检测分析。

方法

从低级别物种秀丽隐杆线虫基因组中选取一段保守序列，利用BLAST检索PLACON序列的特异性，设计成为新型血浆外参照。以恶性肿瘤患者血浆样品及癌细胞株上清液，利用实时荧光定量PCR法，在血浆或细胞上清液中分别以PLACON、β-actin、GAPDH、U6为参照，检测血浆mRNA、miRNA和细胞上清液mRNA并进行对比分析。

结果

BLAST检索出PLACON序列具有较好的特异性，与人类基因组无交叉性。熔解曲线均显示RT-qPCR反应特异性较高，PLAC

ON在血浆中mRNA水平扩增的循环阈值(cycle threshold，CT)均显著低于GAPDH、β-actin(

0.01)，在血浆中miRNA水平扩增的CT值显著低于U6(

0.01)。PLACON在癌细胞上清中进行mRNA扩增实验的验证，得到相似的结果。

结论

PLACON序列特异性好，在血浆中比内参照更有优势，稳定性更高，扩增效率更高，可用于血浆mRNA、miRNA的定量检测。

Abstract

Objective

To design a novel plasma external reference (PLACON) to detect and analyze human plasma mRNA and miRNA.

Methods

A conserved sequence was selected from the genome of the low-level species

Caenorhabditis elegans

. It was designed as a novel plasma external reference and BLAST was used to detect the specificity of PLACON sequences. Plasma samples of patients with malignant tumors and cancer cell supernatants were collected. And PLACON

β-actin

GAPDH

and U6 which were added to the plasma or cell supernatant respectively

were used for amplification comparison to detect plasma mRNA

miRNA and cell supernatant mRNA

by quantitative real-time PCR (RT-qPCR).

Results

BLAST detected that the PLACON sequence had good specificity and no cross-correlation with the human genome. Melting curves showed high specificity in RT-qPCR reaction. The cycle threshold values of mRNA levels were significantly lower in plasma of PLACON group than the GAPDH and β-actin groups (

0.01). The cycle threshold (CT) value of miRNA level amplification in plasma in PLACON group was significantly lower than that in U6 group (

0.01). PLACON was validated in mRNA amplification experiments in cancer cell supernatants

and similar results were obtained.

Conclusion

PLACON sequence is specific

and shows better amplification advantage as internal reference in plasma

with high stability and efficient amplification. It can be used for quantitative detection of plasma mRNA and miRNA.

关键词

Keywords

references

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