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Published on: March 13, 2018
CRISPR-coupled triple cascade amplification for simultaneous lateral flow detection of Mycoplasma pneumoniae and H1N1
Ruyue Wei1, Shuqi Wang1, Yingbo Pan1
1College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Institute of Molecular and Nano Science, Shandong Normal University, Jinan 250014, P. R. China. lina@sdnu.edu.cn.
Abstract:
We developed a CRISPR-coupled triple cascade system integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a, and catalytic hairpin assembly (CHA) for simultaneous lateral flow detection of Mycoplasma pneumoniae and H1N1 in saliva samples, achieving a LOD of 10 aM for H1N1 RNA and 25 aM for MP DNA on a single LFA.
Insights
This study presents a novel CRISPR-based system for detecting Mycoplasma pneumoniae (MP) and H1N1 in saliva. The assay achieves highly sensitive, simultaneous detection of both pathogens on a single lateral flow assay (LFA) strip.
Area of Science:
- Biotechnology
- Molecular Diagnostics
- Infectious Disease Detection
Background:
- Accurate and rapid detection of respiratory pathogens like Mycoplasma pneumoniae (MP) and H1N1 is crucial for timely treatment and public health.
- Current diagnostic methods may lack the sensitivity or multiplexing capability for simultaneous detection of multiple targets from clinical samples like saliva.
Purpose of the Study:
- To develop a highly sensitive and specific CRISPR-coupled diagnostic platform for the simultaneous detection of MP DNA and H1N1 RNA.
- To integrate recombinase polymerase amplification (RPA), CRISPR/Cas12a, and catalytic hairpin assembly (CHA) into a single assay for enhanced detection efficiency.
Main Methods:
- A novel triple cascade system was designed, combining RPA for nucleic acid amplification, CRISPR/Cas12a for specific target recognition, and CHA for signal amplification.
- The system was optimized for simultaneous detection of both MP DNA and H1N1 RNA.
- Lateral flow assay (LFA) strips were utilized for visual and rapid readout of the detection results.
Main Results:
- The developed CRISPR-coupled triple cascade system demonstrated simultaneous detection of both Mycoplasma pneumoniae and H1N1.
- Achieved a limit of detection (LOD) as low as 10 attomolar (aM) for H1N1 RNA and 25 aM for MP DNA.
- The assay was successfully validated using saliva samples, indicating its potential for clinical application.
Conclusions:
- The integrated RPA-CRISPR/Cas12a-CHA system offers a sensitive and specific platform for simultaneous pathogen detection.
- This novel assay provides a promising tool for rapid, point-of-care diagnosis of common respiratory infections.
- The high sensitivity and multiplexing capability make it suitable for early detection of MP and H1N1 in saliva.
