Programmable <i>Clostridium perfringens</i> Argonaute-Based, One-Pot Assay for the Multiplex Detection of miRNAs
Qiuyuan Lin, Yuanwei Cao, Guobin Han, Wen Sun, Wenhao Weng, Hui Chen, Haoyi Wang, Jilie Kong
Abstract
Assays for the molecular detection of miRNAs are typically constrained by the level of multiplexing, especially in a single tube. Here, we report a general and programmable diagnostic platform by combining mesophilic Clostridium perfringens Argonaute (CpAgo) with exponential isothermal amplification (EXPAR), which is a dual-signal amplification strategy, allowing for the rapid and sensitive detection of multiple miRNAs with single-nucleotide discrimination in one pot. The C pAgo-based O ne- P ot (COP) assay achieved a limit of detection of 1 zM miRNA within 30 min of turnaround time and a wide concentration range. This COP assay was applied to simultaneously detect four miRNAs in a single tube from clinical serum samples, showing superior analytical performance in distinguishing colorectal cancer patients from healthy individuals. This programmable, one-pot, multiplex, rapid, and specific strategy offers great promise in scientific research and clinical applications.