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Visual Detection of Multiple Nucleic Acids in a Capillary Array
Published on: November 15, 2017
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Multiplex Digital Nucleic Acid Analysis by a LAMP-Argonaute Coupling Assay via a Parallel Droplet Fusion SlipChip
Yang Luo1, Xingyu Ye2, Nan Shen3
1School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, China.
Analytical Chemistry
|January 15, 2025
Summary
This study introduces a novel multiplex digital nucleic acid analysis (NAA) method using a thermophilic Argonaute protein (Ago) for sensitive detection of multiple viral targets. The system achieves rapid and precise quantification of respiratory viruses like SARS-CoV-2 in clinical samples.
Area of Science:
- Biotechnology
- Molecular Diagnostics
- Bioanalytical Chemistry
Background:
- Multiplex digital nucleic acid analysis (NAA) offers precise quantification of multiple nucleic acids with single-molecule sensitivity, crucial for research and diagnostics.
- While nucleic acid-guided endonucleases show promise for digital NAA, multiplexing remains challenging.
- Thermophilic Argonaute protein (Ago) enables simultaneous targeting of multiple sequences by one enzyme, presenting an opportunity for multiplex detection.
Purpose of the Study:
- To develop a multiplex digital NAA system by integrating nucleic acid amplification with Ago-specific detection.
- To overcome the challenges of multiplex digital NAA using endonucleases.
- To achieve sensitive and specific quantification of multiple nucleic acid targets.
Main Methods:
- Developed a multiplex digital NAA system coupling nucleic acid amplification and Ago-specific detection.
- Utilized parallel droplet fusion facilitated by a SlipChip for generating droplets for multiplex digital loop-mediated isothermal amplification (LAMP).
- Incorporated Ago reagents for parallel mixing and reactions, generating distinct digital fluorescence signals (FAM, ROX, Cy5) for each target.
Main Results:
- Successfully performed multiplex digital loop-mediated isothermal amplification (LAMP) and Ago-specific detection.
- Achieved three distinct digital fluorescence signals (FAM, ROX, Cy5) corresponding to specific target sequences.
- Quantified viral loads of respiratory viruses, including influenza A, influenza B, and SARS-CoV-2, within 60 minutes.
- Analyzed viral loads in 34 clinical samples using the digital LAMP-Ago assay.
Conclusions:
- The developed system provides a sensitive and specific multiplex digital NAA for precise nucleic acid quantification.
- This method demonstrates significant potential for rapid diagnostics and life science research.
- The SlipChip-based digital LAMP-Ago assay offers a robust platform for multiplexed molecular detection.
