Integrated CRISPR-Cas Detection System for Digital Nucleic Acid Amplification
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Solution Overview
Problem
Current nucleic acid testing methods using digital nucleic acid amplification and CRISPR-Cas technology face integration challenges and aerosol contamination issues, leading to cumbersome operations and inaccurate results.
Innovation Solution
An integrated detection system combining droplet digital nucleic acid amplification and CRISPR-Cas technology, utilizing an integrated reaction chip with temperature control, light source, and optical signal detection, where nucleic acid amplification and CRISPR-Cas reagents are divided into microdroplets for precise amplification and detection, avoiding cross-contamination and simplifying operations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple separate instruments (droplet generator, amplification instrument, detector) are used for digital nucleic acid amplification testing, then the detection function is comprehensive, but the device complexity increases and operations become cumbersome
Solution Approach 1:
The patent integrates droplet generation, nucleic acid amplification, and fluorescence detection functions into a single integrated instrument. The device combines a droplet generator module, thermal cycling module for amplification, and optical detection module, eliminating the need for multiple separate instruments and reducing operational complexity while maintaining comprehensive detection capabilities
Solution Approach 2:
The integrated instrument is designed to perform multiple functions: generating microdroplets, performing thermal cycling for nucleic acid amplification, and detecting fluorescence signals. This multi-functional design allows a single device to replace multiple specialized instruments, simplifying the workflow and reducing device complexity
2Ease of operation
If CRISPR-Cas detection reagents are added after amplification by opening the cap, then the reagents can be added, but aerosol contamination occurs causing false positives
Solution Approach 1:
The patent integrates the CRISPR-Cas detection reagent addition step into the closed microdroplet system. The reagents are introduced through the sealed reaction chamber without opening the cap, using a liquid addition module that maintains the closed system integrity, thereby preventing aerosol contamination while enabling reagent addition
Solution Approach 2:
The patent uses a liquid addition module as an intermediary mechanism to introduce CRISPR-Cas reagents into the closed reaction system. This intermediary allows reagent addition without breaking the sealed environment, preventing contamination while maintaining ease of operation
3Ease of operation
If fluorescent dyes are used for detection, then the detection is simple, but the specificity is poor and cannot distinguish specific amplification products
Solution Approach 1:
The patent introduces CRISPR-Cas system components (guide RNA and Cas12a enzyme) as intermediaries between the amplification process and fluorescence detection. The guide RNA specifically binds to the target sequence, and Cas12a cleaves the fluorescent probe only when the target is present, providing high specificity while maintaining fluorescence-based detection simplicity
Solution Approach 2:
The patent uses site-specific fluorescence quenching and activation. The fluorescent probe is designed with a specific structure where fluorescence is quenched until Cas12a cleaves it upon target recognition. This local quality change at the molecular level provides high specificity while maintaining detection simplicity
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 enables absolute quantitative and high-specificity testing, integrating droplet generation, amplification, and fluorescence detection on a single chip, reducing contamination risks and simplifying operations while maintaining the sensitivity and specificity of CRISPR-Cas systems.
Implementation Method 1
the CRISPR-Cas12a system may identify and capture the target strand according to the designed gRNA (guide Ribonucleic Acid), and its DNA (Deoxyribonucleic Acid) enzyme cleavage activity is activated
Implementation Method 2
efficiently cleaving the single-stranded fluorescent probe in the system, so as to achieve specificity testing of target DNA
Implementation Method 3
nucleic acid amplification and CRISPR-Cas reagents are divided into microdroplets for precise amplification and detection
Implementation Method 4
An integrated detection system combining droplet digital nucleic acid amplification and CRISPR-Cas technology, utilizing an integrated reaction chip with temperature control
Implementation Method 5
optical signal detection, where nucleic acid amplification and CRISPR-Cas reagents are divided into microdroplets for precise amplification and detection
Data Source
AI summary
Disclosed in the present invention are a digital nucleic acid amplification testing method and an integrated detection system based on CRISPR-Cas technology. The integrated detection system comprises an integrated reaction chip, a temperature control module, a light source and an optical signal detector. The method comprises: uniformly dividing a nucleic acid amplification reagent into amplification micro-droplets, then mixing the amplification micro-droplets after digital nucleic acid amplification with detection micro-droplets containing CRISPR-Cas detection reagent to perform a CRISPR reaction, and when the reaction is finished, detecting an optical signal to realize high-specificity testing of a target object, and the concentration or copy number of nucleic acid molecules in a sample to be tested is also obtained, and high-sensitivity absolute quantitative testing of a target object is realized.