One-pot Ago Isothermal Assay
This technology uses a thermophilic Argonaute protein to rapidly and accurately detect small amounts of DNA or RNA, including mutations, from various sources with high sensitivity, specificity, and multiplex capability, making it useful for non-invasive disease diagnostics.
The Problem
Despite significant advances, current approaches for detecting cell-free DNA (cfDNA) and cell-free RNA (cfRNA) face several limitations. Techniques such as quantitative real-time PCR (qRT-PCR), digital PCR, and CRISPR-Cas-based assays often require complex workflows, expensive reagents, and specialized equipment, making them less accessible in resource-limited settings. Furthermore, many of these methods are limited in their ability to simultaneously detect multiple targets (multiplexing) and may struggle with distinguishing between highly similar sequences, such as single nucleotide variants. Sensitivity and specificity can also be compromised by background noise or off-target effects, while the need for precise thermal cycling or enzymatic steps can introduce variability and increase turnaround time. These challenges highlight the need for improved solutions that combine simplicity, speed, multiplex capability, and high analytical performance.
The Solution
This technology is a protein-based assay designed for the quantitative detection of small single-stranded RNAs (ssRNAs), single-stranded DNAs (ssDNAs), and double-stranded DNAs (dsDNAs) in cell-free samples. The method generates a fluorescence signal, in the presence of a nucleic acid of interest, enabling sensitive and specific detection. The assay is highly versatile, capable of identifying a wide range of DNA and RNA targets, including those from pathogens (such as viruses and bacteria) and host-derived molecules, like microRNAs (miRNAs). Its design supports true multiplex detection, allowing simultaneous analysis of multiple targets, and can even detect single nucleotide mutations.
The Opportunity
What differentiates this technology is its unique combination of features that address the limitations of existing cfDNA and cfRNA detection methods. Unlike conventional qRT-PCR, digital PCR, and CRISPR-Cas assays, this approach offers a rapid and straightforward operational procedure, high sensitivity and specificity, and robust performance at a low cost.
