CRISPR Coronavirus Diagnostics With Single-Reaction Isothermal Detection
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Solution Overview
Problem
Current methods for detecting nucleic acids, such as those for the novel coronavirus, face challenges in achieving high sensitivity, specificity, and speed, particularly in field-deployable tests, which are crucial for rapid diagnosis and outbreak management.
Innovation Solution
A single-reaction composition using thermostable CRISPR Cas proteins with collateral activity, combined with isothermal amplification reagents like LAMP, enables extraction-free nucleic acid detection, allowing for rapid and specific identification of target sequences without the need for separate extraction steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional nucleic acid detection methods are used, then detection sensitivity and specificity can be achieved, but the detection time is extended to four hours and separate extraction steps are required
Solution Approach 1:
The patent combines the extraction step and amplification step into a single reaction mixture, eliminating the need for separate extraction and purification steps. The extraction buffer components are integrated directly with the amplification reagents, allowing nucleic acid extraction and isothermal amplification to occur simultaneously in one tube, thereby reducing detection time from four hours to 30-120 minutes while maintaining detection sensitivity and specificity
Solution Approach 2:
The patent performs preliminary optimization of the extraction buffer composition to ensure it is compatible with subsequent amplification reactions. The extraction buffer is pre-formulated with specific components (chelating agents, detergents, salts) that facilitate both nucleic acid extraction and serve as compatible conditions for isothermal amplification, eliminating the need for post-extraction processing steps
2Measurement precision
If conventional nucleic acid detection methods are used, then detection accuracy is maintained, but the procedure complexity increases due to multiple separate steps
Solution Approach 1:
The patent merges multiple procedural steps (extraction, purification, amplification) into a single integrated reaction protocol. The extraction buffer contains all necessary components for nucleic acid release and stabilization, and these same components are compatible with the amplification reaction, allowing the entire process to be performed in one tube without intermediate purification steps, thereby simplifying the procedure while maintaining detection accuracy
Solution Approach 2:
The extraction buffer is designed to perform multiple functions simultaneously: it lyses viral particles, releases nucleic acids, stabilizes the extracted nucleic acids, and provides compatible conditions for the subsequent amplification reaction. This multi-functional buffer eliminates the need for separate extraction and amplification buffers, reducing procedural complexity while maintaining detection accuracy
3Speed
If field-deployable rapid tests are developed, then detection speed is improved, but detection sensitivity and specificity are compromised
Solution Approach 1:
The patent changes the temperature parameter from conventional PCR cycling temperatures to a constant isothermal temperature (50-65°C) suitable for field deployment. The extraction buffer is specifically optimized to maintain nucleic acid stability and reaction efficiency at these isothermal conditions, enabling rapid detection (30-120 minutes) with high sensitivity and specificity using simple heating blocks or water baths available in field settings
Solution Approach 2:
The patent replaces complex automated extraction systems with a simple chemical extraction approach using a pre-formulated buffer that requires no mechanical manipulation. The buffer chemically lyses viral particles and releases nucleic acids through chelation of divalent cations and disruption of viral envelopes, eliminating the need for centrifugation, filtration, or column-based purification steps, thereby enabling rapid field deployment while maintaining detection sensitivity and specificity
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
This approach provides rapid, sensitive, and specific detection of nucleic acids, capable of generating results within 30-120 minutes, with high sensitivity and specificity, suitable for point-of-care diagnostics and effective in detecting low concentrations of viral targets.
Implementation Method 1
the Cas protein exhibits collateral nuclease activity and cleaves the polynucleotide component of the detection construct once activated by the target sequence
Implementation Method 2
at least one guide polynucleotide comprising a sequence capable of binding a target polynucleotide and designed to form a complex with the one or more Cas proteins
Implementation Method 3
isothermal amplification reagents
Data Source
AI summary
Systems and methods for rapid diagnostics related to the use of CRISPR effector systems and optimized guide sequences for detection of coronavirus, including multiplex lateral flow diagnostic devices and methods of use, are provided.