<i>Thermus thermophilus</i> Argonaute-Based Isothermal Amplification Assay for Ultrasensitive and Specific RNA Detection

Abstract

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 <i>Thermus thermophilus</i> Argonaute-based thermostable exponential amplification reaction (<i>Tt</i>AgoEAR), 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 <i>Tt</i>AgoEAR 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 <i>Tt</i>AgoEAR is easily adaptable to a lateral-flow-based readout. These results demonstrate that <i>Tt</i>AgoEAR has great potential to facilitate reliable and easy RNA detection in point-of-care diagnosis and field analysis.