CRISPR-Based Nucleic Acid Detection for Pathogenic Mutants
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting pathogenic mutants, such as SARS-CoV-2, are limited by low applicability, sensitivity, long time consumption, high cost, and dependence on equipment and instruments, making them inefficient for rapid and accurate detection.
Innovation Solution
An instant nucleic acid detection method using the CRISPR process combined with colloidal gold test paper or fluorescence signal detection, which involves sample nucleic acid extraction, crRNA design for specific Cas proteins, and subsequent amplification using RPA or LAMP reactions to enhance sensitivity and specificity, allowing for rapid and accurate detection of pathogenic mutants without requiring complex equipment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If whole genome sequencing is used to detect pathogenic mutants, then detection accuracy is improved, but time consumption and cost increase significantly
Solution Approach 1:
The patent extracts and detects only specific mutation points of interest from the pathogen genome using targeted primers and probes, rather than sequencing the entire genome. This selective extraction approach maintains detection accuracy for specific mutants while dramatically reducing time and resource requirements compared to whole genome sequencing
Solution Approach 2:
The detection method segments the pathogen genome into specific regions of interest (mutation points) and designs targeted detection reactions for each. By dividing the detection task into focused segments rather than analyzing the complete genome, the method achieves rapid and accurate detection of specific mutants without the time burden of comprehensive sequencing
2Measurement precision
If RT-qPCR is used for pathogen detection, then detection sensitivity is improved, but applicability to genetic typing of mutants is limited
Solution Approach 1:
The patent employs dynamic probe design where fluorescent probes are specifically tailored to match different mutant sequences. The detection system can be dynamically reconfigured by changing the probe sequences to detect various mutants, maintaining high detection sensitivity while achieving broad applicability to genetic typing through flexible, adaptable reagent design
Solution Approach 2:
The method applies local quality by designing detection primers and probes with specific sequences that match particular mutation points. Each detection reaction is optimized for its specific target region, enabling sensitive detection of specific mutants while the overall system achieves versatility through multiple specialized detection assays
3Productivity
If multiplex qPCR is used to distinguish SARS-CoV-2 mutants, then detection speed is improved, but sensitivity to unit point mutation decreases
Solution Approach 1:
The patent performs preliminary amplification of the target region using RPA or LAMP reactions before detection. This pre-amplification step enriches the target DNA sequences, including those with single point mutations, to sufficient levels for sensitive detection. The preliminary action ensures that even unit point mutations are adequately represented and detectable in the subsequent CRISPR-based detection step
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 method significantly increases detection sensitivity and specificity, enabling rapid and accurate identification of pathogenic mutants, including SARS-CoV-2 variants, and allows for visual reading of results without relying on instruments, making it suitable for self-inspection and use in hospitals.
Implementation Method 1
designing crRNA for a CRISPR process, selecting Cas9 nuclease, Cas13 nuclease or Cas12a nuclease as a Cas protein used in the CRISPR process
Implementation Method 2
crRNA (guide RNA), which is capable of guiding the Cas protein specifically binding to a target DNA fragment
Implementation Method 3
detecting the sample nucleic acids by using the CRISPR process combined with a colloidal gold test paper method
Implementation Method 4
detecting the sample nucleic acids by using the CRISPR process combined with a fluorescence signal detection method
Implementation Method 5
subsequent amplification using RPA or LAMP reactions to enhance sensitivity and specificity
Data Source
AI summary
The present disclosure discloses an instant nucleic acid detection method and detection kit for detecting pathogenic mutants, belongs to the technical field of biological detection, includes the following steps: Step 1): collecting samples, and extracting sample nucleic acids; Step 2): designing crRNA used in a CRISPR process, selecting Cas9 nuclease, Cas13 nuclease or Cas12a nuclease; and Step 3): detecting the sample nucleic acids by using the CRISPR process combined with a colloidal gold test paper method or a fluorescence signal detection method.