Department of Chemical and Biomolecular Engineering (BK21+ Program), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.
Nanoscale. 2021 Jun 24;13(24):10785-10791. doi: 10.1039/d1nr00564b.
Nucleic acid sequence-based amplification (NASBA) is a transcription-based isothermal amplification technique especially designed for the detection of RNA targets. The NASBA basically relies on the linear production of T7 RNA promoter-containing double-stranded DNA (T7DNA), and thus the final amplification efficiency is not sufficiently high enough to achieve ultrasensitive detection. We herein ingeniously integrate a nicking and extension chain reaction system into the NASBA to establish an ultrasensitive version of NASBA, termed Nicking and Extension chain reaction System-Based Amplification (NESBA). By employing a NESBA primer set designed to contain an additional nicking site at the 5' end of a NASBA primer set, the T7DNA is exponentially amplified through continuously repeated nicking and extension chain reaction by the combined activities of nicking endonuclease (NE) and reverse transcriptase (RT). As a consequence, a much larger number of RNA amplicons would be produced through the transcription of the amplified T7DNA, greatly enhancing the final fluorescence signal from the molecular beacon (MB) probe binding to the RNA amplicon. Based on this unique design principle, we successfully identified the target respiratory syncytial virus A (RSV A) genomic RNA (gRNA) down to 1 aM under isothermal conditions, which is 100-fold more sensitive than regular NASBA.
基于核酸序列的扩增（NASBA）是一种转录物等温扩增技术，专门设计用于检测 RNA 靶标。NASBA 基本上依赖于 T7 RNA 启动子包含的双链 DNA（T7DNA）的线性产生，因此最终的扩增效率不够高，无法实现超灵敏检测。在此，我们巧妙地将缺口和延伸链反应系统整合到 NASBA 中，建立了一种超灵敏的 NASBA 版本，称为缺口和延伸链反应系统扩增（NESBA）。通过使用设计的 NESBA 引物组在 NASBA 引物组的 5'端包含额外的缺口位点，通过切口内切酶（NE）和逆转录酶（RT）的联合活性，T7DNA 通过连续重复的切口和延伸链反应呈指数扩增。结果，通过扩增的 T7DNA 的转录会产生更多数量的 RNA 扩增子，从而大大增强了与 RNA 扩增子结合的分子信标（MB）探针的最终荧光信号。基于这种独特的设计原理，我们成功地在等温条件下将目标呼吸道合胞病毒 A（RSV A）基因组 RNA（gRNA）检测到低至 1 aM 的水平，比常规 NASBA 灵敏 100 倍。
