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Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases
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Dynamic Aqueous Multiphase Reaction System for One-Pot CRISPR-Cas12a-Based Ultrasensitive and Quantitative Molecular
Kun Yin1, Xiong Ding1, Ziyue Li1
1Department of Biomedical Engineering, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06030, United States.
Analytical Chemistry
|May 12, 2020
Summary
A new dynamic aqueous multiphase reaction (DAMR) system enables simple, sensitive, and quantitative CRISPR-Cas12a molecular diagnostics. This one-pot method detects human papillomavirus (HPV) DNA in under an hour, even from raw plasma samples.
Area of Science:
- Molecular Diagnostics
- Biotechnology
- CRISPR-Cas Systems
Background:
- CRISPR-Cas technology has revolutionized nucleic acid-based molecular diagnostics.
- Existing CRISPR-Cas biosensing methods often lack quantitative detection and require manual, multi-step operations.
- There is a need for simplified, sensitive, and quantitative diagnostic platforms.
Purpose of the Study:
- To develop a novel dynamic aqueous multiphase reaction (DAMR) system for one-pot CRISPR-Cas12a molecular diagnosis.
- To achieve simple, sensitive, and quantitative detection of nucleic acids.
- To demonstrate the system's utility for detecting human papillomavirus (HPV) DNA and its potential for point-of-care applications.
Main Methods:
- Developed a dynamic aqueous multiphase reaction (DAMR) system utilizing sucrose density gradients for phase separation.
- Integrated recombinase polymerase amplification (RPA) and CRISPR-Cas12a fluorescent detection within the DAMR system.
- Employed a 3D-printed microfluidic device for sample handling and multiplex detection.
Main Results:
- The DAMR system enabled quantitative detection of HPV 16 and 18 DNAs with high sensitivity (10 and 100 copies, respectively) in under 1 hour.
- Successfully performed multiplex detection of HPV16/18 in clinical human swab samples.
- Demonstrated direct amplification and detection of target DNA from human plasma without complex pretreatment.
Conclusions:
- The DAMR system offers a simple, sensitive, and quantitative approach for CRISPR-Cas12a-based molecular diagnostics.
- The developed system successfully detected HPV DNA in clinical samples and raw plasma, highlighting its robustness.
- The DAMR system shows significant potential for the development of next-generation point-of-care molecular diagnostic tools.
