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Thermus thermophilus Argonaute-Based Isothermal Amplification Assay for Ultrasensitive and Specific RNA Detection
Analytical Chemistry ( IF 6.7 ) Pub Date : 2023-05-17 , DOI: 10.1021/acs.analchem.3c00599
Changjing Yuan 1, 2 , Jie Fang 1, 3 , Weiling Fu 1
Analytical Chemistry ( IF 6.7 ) Pub Date : 2023-05-17 , DOI: 10.1021/acs.analchem.3c00599
Changjing Yuan 1, 2 , Jie Fang 1, 3 , Weiling Fu 1
Affiliation
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Recent advances in prokaryotic Argonaute proteins (pAgos) as potential genome-editing tools have provided new insights into the development of pAgos-based nucleic acid detection platforms. However, pAgos-based isothermal detection remains challenging. Here, we report a true isothermal amplification strategy, termed Thermus thermophilus Argonaute-based thermostable exponential amplification reaction (TtAgoEAR), to detect RNA with ultrasensitivity and single-nucleotide resolution at a constant temperature of 66 °C. We demonstrate the reliable detection of lncRNA, mRNA, and virus RNA with attomolar sensitivity and that TtAgoEAR can be applied to detect RNA targets in in cell lines, saliva, and tissues. We utilize this assay to distinguish pancreatic cancer cells carrying the mutation from wild-type cells with as little as 2 ng of RNA material. We also show that TtAgoEAR is easily adaptable to a lateral-flow-based readout. These results demonstrate that TtAgoEAR has great potential to facilitate reliable and easy RNA detection in point-of-care diagnosis and field analysis.
中文翻译:
基于 Thermus thermophilus Argonaute 的等温扩增试验用于超灵敏和特异性 RNA 检测
原核生物 Argonaute 蛋白 (pAgos) 作为潜在的基因组编辑工具的最新进展为基于 pAgos 的核酸检测平台的开发提供了新的见解。然而,基于 pAgos 的等温检测仍然具有挑战性。在这里,我们报告了一种真正的等温扩增策略,称为基于Thermus thermophilus Argonaute 的热稳定指数扩增反应 ( Tt AgoEAR),以在 66 °C 的恒定温度下检测具有超灵敏度和单核苷酸分辨率的 RNA。我们证明了 lncRNA、mRNA 和病毒 RNA 的可靠检测具有阿托摩尔灵敏度,并且TtAgoEAR 可用于检测细胞系、唾液和组织中的 RNA 靶标。我们利用这种测定法来区分携带突变的胰腺癌细胞和具有低至 2 ng RNA 材料的野生型细胞。我们还表明,Tt AgoEAR 很容易适应基于侧流的读数。这些结果表明,Tt AgoEAR 在促进即时诊断和现场分析中可靠且简单的 RNA 检测方面具有巨大潜力。
更新日期:2023-05-17
中文翻译:
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基于 Thermus thermophilus Argonaute 的等温扩增试验用于超灵敏和特异性 RNA 检测
原核生物 Argonaute 蛋白 (pAgos) 作为潜在的基因组编辑工具的最新进展为基于 pAgos 的核酸检测平台的开发提供了新的见解。然而,基于 pAgos 的等温检测仍然具有挑战性。在这里,我们报告了一种真正的等温扩增策略,称为基于Thermus thermophilus Argonaute 的热稳定指数扩增反应 ( Tt AgoEAR),以在 66 °C 的恒定温度下检测具有超灵敏度和单核苷酸分辨率的 RNA。我们证明了 lncRNA、mRNA 和病毒 RNA 的可靠检测具有阿托摩尔灵敏度,并且TtAgoEAR 可用于检测细胞系、唾液和组织中的 RNA 靶标。我们利用这种测定法来区分携带突变的胰腺癌细胞和具有低至 2 ng RNA 材料的野生型细胞。我们还表明,Tt AgoEAR 很容易适应基于侧流的读数。这些结果表明,Tt AgoEAR 在促进即时诊断和现场分析中可靠且简单的 RNA 检测方面具有巨大潜力。