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A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System
Published on: April 6, 2021
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Multiplexed lateral flow assay integrated with orthogonal CRISPR-Cas system for SARS-CoV-2 detection
Gaoxing Su1, Min Zhu1, Diyuan Li1
1School of Pharmacy, Nantong University, Nantong, Jiangsu 226001, China.
Sensors and Actuators. B, Chemical
|August 29, 2022
Summary
A novel orthogonal CRISPR-Cas multiplexed lateral flow assay (OC-MLFA) enables sensitive and accurate SARS-CoV-2 detection. This field-deployable platform offers reliable point-of-care testing for infectious diseases.
Area of Science:
- Molecular Biology
- Biotechnology
- Diagnostics
Background:
- Point-of-care testing (POCT) requires efficient and reliable field-deployable platforms for multiplexed gene detection.
- Current methods for detecting pathogens like SARS-CoV-2 can be limited in speed, sensitivity, or multiplexing capability.
Purpose of the Study:
- To develop an orthogonal CRISPR-Cas-mediated multiplexed lateral flow assay (OC-MLFA) for sensitive and accurate SARS-CoV-2 genome detection.
- To leverage the distinct activation and cleavage properties of Cas12a and Cas13a enzymes for a dual-channel signal readout.
- To validate the OC-MLFA's performance using clinical nasopharyngeal swab samples.
Main Methods:
- An orthogonal CRISPR-Cas system utilizing Cas12a and Cas13a was designed for multiplexed detection.
- Lateral flow strips with two target lines were engineered to accommodate the orthogonal CRISPR system, minimizing channel interference.
- Reverse transcription recombinase polymerase amplification (RT-RPA) was employed for preamplification, followed by Cas enzyme-mediated trans-cleavage for signal generation.
Main Results:
- The OC-MLFA achieved a high sensitivity of 10 copies per test (30 μL) due to efficient preamplification and Cas trans-cleavage.
- The assay demonstrated excellent accuracy, with 76 out of 76 clinical nasopharyngeal swab samples correctly identified.
- The orthogonal design effectively eliminated interference between the Cas12a and Cas13a detection channels.
Conclusions:
- The developed OC-MLFA provides a highly accurate and efficient method for simultaneous multiplexed gene detection with a simple colorimetric readout.
- This field-deployable assay shows significant promise for reliable point-of-care testing applications, particularly for infectious disease surveillance.
- The orthogonal CRISPR-Cas strategy offers a robust platform for advancing multiplexed diagnostics in resource-limited settings.
