Institute of Biochemistry and Genetics, Ufa Federal Research Center, Russian Academy of Sciences, Ufa, 450054, Bashkortostan, Russia.
Biochemistry (Mosc). 2023 May;88(5):679-686. doi: 10.1134/S0006297923050103.
Detection of specific RNA targets via amplification-mediated techniques is widely used in fundamental studies and medicine due to essential role of RNA in transfer of genetic information and development of diseases. Here, we report on an approach for detection of RNA targets based on the particular type of isothermal amplification, namely, reaction of nucleic acid multimerization. The proposed technique requires only a single DNA polymerase possessing reverse transcriptase, DNA-dependent DNA polymerase, and strand-displacement activities. Reaction conditions that lead to efficient detection of the target RNAs through multimerization mechanism were determined. The approach was verified by using genetic material of the SARS-CoV-2 coronavirus as a model viral RNA. Reaction of multimerization allowed to differentiate the SARS-CoV-2 RNA-positive samples from the SARS-CoV-2 negative samples with high reliability. The proposed technique allows detection of RNA even in the samples, which were subjected to multiple freezing-thawing cycles.
基于扩增介导的技术检测特定的 RNA 靶标由于 RNA 在遗传信息传递和疾病发生中的重要作用，在基础研究和医学中得到了广泛应用。在这里，我们报告了一种基于核酸多聚化的特定类型的等温扩增反应检测 RNA 靶标的方法。所提出的技术仅需要一种具有逆转录酶、DNA 依赖性 DNA 聚合酶和链置换活性的单一 DNA 聚合酶。确定了通过多聚化机制高效检测靶 RNA 的反应条件。该方法通过使用 SARS-CoV-2 冠状病毒的遗传物质作为模型病毒 RNA 进行了验证。多聚化反应能够可靠地区分 SARS-CoV-2 RNA 阳性样本和 SARS-CoV-2 阴性样本。该技术甚至可以检测经过多次冻融循环的样本中的 RNA。
