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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
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放大无偏差序列-通用指数级放大反应反应

Xinrong Yan1, Qingyuan Wang2, Peiru Yang1

  • 1State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, P. R. China.

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此摘要是机器生成的。

这项研究引入了序列通用指数放大反应 (SG-EXPAR),这是一种用于核酸检测的新方法. SG-EXPAR克服了放大偏差,为各种目标实现了高度敏感和一致的结果.

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科学领域:

  • 分子生物学分子生物学
  • 生物技术是生物技术.
  • 核酸扩大核酸扩大

背景情况:

  • 同热指数放大反应 (EXPAR) 提供了快速的核酸检测,但患有依赖序列的放大偏差.
  • 这种偏差是由55°C的模板二次结构引起的,导致从aM到nM的可变检测极限 (LOD).
  • 现有的方法在不同的目标序列中缺乏一致的性能.

研究的目的:

  • 开发一个序列通用的指数放大反应 (SG-EXPAR),消除放大偏差.
  • 为了在各种目标上实现一致和高度灵敏的核酸检测.
  • 为了使可靠的护理点核酸测定.

主要方法:

  • 为了使SG-EXPAR在更高的温度 (60-70°C) 上运行,采用了热友的缩酶,消除了模板的二次结构.
  • 优化锁定核酸和模板设计,以提高触发器/模板结合的概率.
  • 开发了一个自动化设计平台,用于为任何序列创建最佳的SG-EXPAR模板.

主要成果:

  • 通过一般的fM以下LOD实现了序列通用放大,消除了依赖序列的偏差.
  • 在没有序列查的情况下,在1 fM级别量化微RNA,SARS-CoV-2,病毒和HPV B19方面表现出强大的性能.
  • 验证了该方法的效率和广泛适用于各种核酸点.

结论:

  • SG-EXPAR通过消除依赖序列的放大偏差来克服传统EXPAR的局限性.
  • 开发的方法提供了对各种核酸,包括病原体的高度敏感和一致的检测.
  • SG-EXPAR显著扩展EXPAR应用,并支持开发可靠的临床诊断工具.