iSCAN SARS-CoV-2 Detection via RT-LAMP and CRISPR-Cas12
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Solution Overview
Problem
Current methods for detecting SARS-CoV-2, such as RT-qPCR, are limited by the need for trained personnel, sophisticated infrastructure, and are not suitable for large-scale point-of-care diagnostics, especially during emergencies, and existing isothermal amplification methods like RT-LAMP face challenges with specificity and false positives.
Innovation Solution
The development of the iSCAN method, which combines RT-LAMP with CRISPR-Cas12 or Cas13 systems for rapid, specific, and sensitive detection of SARS-CoV-2 nucleic acids, using simple equipment and a colorimetric reaction for easy result assessment, allowing for field-deployable and accurate diagnostics.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If RT-qPCR is used for SARS-CoV-2 detection, then sensitivity and specificity are improved, but device complexity and requirement for trained personnel increase
Solution Approach 1:
The diagnostic system is divided into modular components: sample processing module, isothermal amplification module, and detection module. Each module can be independently optimized and deployed, allowing the system to achieve high sensitivity without requiring complex centralized infrastructure. The segmentation enables point-of-care implementation while maintaining RT-qPCR-level detection accuracy.
Solution Approach 2:
A signal amplification intermediary system is introduced between the target nucleic acid and the detection readout. This intermediary uses catalytic hairpin assembly and lateral flow assay technology to amplify signals, enabling sensitive detection without requiring complex instrumentation. The intermediary converts molecular detection into visible signals that can be read without trained personnel.
2Measurement precision
If RT-qPCR is used for SARS-CoV-2 detection, then detection accuracy is improved, but time required for analysis increases
Solution Approach 1:
The system employs periodic thermal cycling combined with isothermal amplification steps. The periodic heating and cooling cycles enable rapid nucleic acid denaturation and primer annealing, significantly reducing the total analysis time while maintaining detection accuracy through multiple amplification cycles that ensure complete target detection.
Solution Approach 2:
The system changes the temperature parameter from periodic cycling (RT-qPCR) to isothermal constant temperature (RT-LAMP). This parameter change enables faster reaction kinetics and eliminates time-consuming thermal cycling steps, reducing analysis time from hours to minutes while maintaining high detection accuracy through optimized isothermal amplification conditions.
3Ease of operation
If isothermal amplification methods like RT-LAMP are used, then ease of operation is improved, but measurement precision deteriorates due to false positives
Solution Approach 1:
The system incorporates control samples and negative controls in each test run to provide feedback on assay performance. The lateral flow assay visually indicates whether the reaction is proceeding correctly, allowing operators to identify and correct false positives immediately. This feedback mechanism maintains high specificity while preserving the ease of operation at point-of-care settings.
Solution Approach 2:
The detection system uses composite reagent formulations that combine multiple functional components in a single buffer system. These composite reagents include specialized primers, nucleotides, and enzymes that work synergistically to achieve both high specificity and ease of operation. The composite formulation eliminates the need for separate reagent steps, simplifying operation while maintaining detection precision.
4Measurement precision
If RT-qPCR is used for large-scale screening, then detection capability is improved, but loss of time for sample transport and processing increases
Solution Approach 1:
The system performs sample processing and nucleic acid extraction in the field before samples reach the detection laboratory. Preliminary actions include RNA stabilization, nucleic acid purification, and quality control, all performed using portable kits. This eliminates time-consuming transport steps and enables rapid detection at the point of care, significantly reducing the overall time loss for large-scale screening operations.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The iSCAN method provides rapid, specific, and sensitive detection of SARS-CoV-2, capable of identifying the virus in under an hour with high accuracy, suitable for large-scale field deployment, and validated using clinical samples, facilitating early detection and isolation of carriers to control the virus spread.
Implementation Method 1
Collateral activation of CRISPR-Cas12 or Cas13 endonuclease activity
Implementation Method 2
CRISPR-Cas12 or Cas13 endonuclease activity
Implementation Method 3
isothermal amplification methods like RT-LAMP
Implementation Method 4
RT-LAMP with CRISPR-Cas12 or Cas13 systems for rapid, specific, and sensitive detection
Data Source
AI summary
Compositions and use of the compositions in methods of detecting SARS-CoV-2 in a sample is disclosed, using RT-LAMP coupled with CRISPR-Cas12, referred to herein as iSCAN (in vitro Scanning of COVID-19-Associated Nucleic acids) is disclosed. iSCAN provides a rapid, specific, accurate, sensitive detection of SARS-CoV-2 in a sample. The iSCAN is 1) rapid, as the RT-LAMP and CRISPR-Cas12/Cas 13 reaction takes less than 1 h; 2) specific, because detection depends on the identification and subsequent cleavage of SARS-CoV-2 genomic sequences by the Cas12 or 13 enzyme; 3) field-deployable, as only simple equipment is required; and 4) easy to use, as the colorimetric reaction coupled to lateral flow immunochromatography makes the assay results easy to assess. The methods include amplifying SARS-CoV-2 in a sample using RT-LAMP and using the RT-LAMP product as a substrate in a CRISPR-Cas12/13 reaction, incorporated with a means of detecting the presence of the SARS-CoV-2 RT-LAMP product.