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Published on: November 30, 2016
Programmable Clostridium perfringens Argonaute-Based, One-Pot Assay for the Multiplex Detection of miRNAs
Qiuyuan Lin1, Yuanwei Cao2, Guobin Han1
1Department of Chemistry, Fudan University, 200438 Shanghai, China.
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 CpAgo-based One-Pot (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.
Insights
This study introduces a novel diagnostic platform for detecting multiple microRNAs (miRNAs) simultaneously. The CpAgo-based One-Pot (COP) assay offers rapid, sensitive, and specific detection, proving effective in distinguishing colorectal cancer patients from healthy individuals.
Area of Science:
- Biotechnology
- Molecular Diagnostics
- Cancer Biomarkers
Background:
- MicroRNA (miRNA) detection assays often face limitations in multiplexing capabilities within a single reaction tube.
- Developing sensitive and specific methods for simultaneous detection of multiple miRNAs is crucial for advanced diagnostics.
Purpose of the Study:
- To develop a general and programmable diagnostic platform for sensitive, single-tube, multiplex miRNA detection.
- To evaluate the performance of the developed assay in distinguishing cancer patients from healthy individuals.
Main Methods:
- Integration of mesophilic *Clostridium perfringens* Argonaute (CpAgo) with exponential isothermal amplification (EXPAR) to create a dual-signal amplification strategy.
- Development of the CpAgo-based One-Pot (COP) assay for simultaneous detection of multiple miRNAs with single-nucleotide discrimination.
- Application of the COP assay to analyze clinical serum samples for miRNA profiling.
Main Results:
- The COP assay demonstrated a limit of detection as low as 1 zM miRNA within a 30-minute timeframe.
- The assay exhibited a wide dynamic range and high specificity, enabling single-nucleotide discrimination.
- Simultaneous detection of four miRNAs in clinical serum samples successfully differentiated colorectal cancer patients from healthy controls.
Conclusions:
- The programmable, one-pot, multiplex COP assay provides a rapid and specific method for miRNA detection.
- This strategy holds significant promise for advancing scientific research and clinical diagnostic applications, particularly in cancer detection.
