<i>Thermus thermophilus</i> Argonaute-Based Isothermal Amplification Assay for Ultrasensitive and Specific RNA Detection
Changjing Yuan, Jie Fang, Weiling Fu
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 Thermus thermophilus Argonaute-based thermostable exponential amplification reaction ( Tt 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 Tt 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 Tt AgoEAR is easily adaptable to a lateral-flow-based readout. These results demonstrate that Tt AgoEAR has great potential to facilitate reliable and easy RNA detection in point-of-care diagnosis and field analysis.