Programmable Clostridium perfringens Argonaute-Based, One-Pot Assay for the Multiplex Detection of miRNAs

Assays for the molecular detection of miRNAs are typicallyconstrainedby the level of multiplexing, especially in a single tube. Here, wereport a general and programmable diagnostic platform by combiningmesophilic Clostridium perfringens Argonaute (CpAgo)with exponential isothermal amplification (EXPAR), which is a dual-signalamplification strategy, allowing for the rapid and sensitive detectionof multiple miRNAs with single-nucleotide discrimination in one pot.The CpAgo-based One-Pot (COP) assay achieved a limit of detection of 1 zMmiRNA within 30 min of turnaround time and a wide concentration range.This COP assay was applied to simultaneously detect four miRNAs ina single tube from clinical serum samples, showing superior analyticalperformance in distinguishing colorectal cancer patients from healthyindividuals. This programmable, one-pot, multiplex, rapid, and specificstrategy offers great promise in scientific research and clinicalapplications.