CRISPR/Cas12a Pathogen Detection via Trans Cleavage
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Solution Overview
Problem
Current methods for detecting pathogenic bacteria in clinical samples, such as those causing severe pneumonia, are time-consuming, prone to false positives due to contamination, and lack specificity, especially for anaerobic bacteria and those difficult to culture, leading to delayed and inaccurate diagnoses.
Innovation Solution
A method utilizing pathogen-specific nucleic acid fragments, identified through whole genome sequencing and CRISPR/Cas12a-based detection, allows for rapid identification of pathogens like Acinetobacter baumannii, Escherichia coli, and others by amplifying and detecting specific DNA sequences using CRISPR/Cas12a's trans cleavage activity, reducing detection time to less than 3 hours.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional colony culture method is used for pathogen detection, then pathogen identification can be achieved through phenotypic and physiological characteristics, but the detection process is time-consuming (requiring one to several days of colony enrichment culture) and may yield negative results for anaerobic pathogenic bacteria or strains hard to be cultured
Solution Approach 1:
The patent replaces the mechanical colony culture system with a molecular biology-based detection system using nucleic acid amplification (PCR) and CRISPR/Cas12a technology. This substitution eliminates the need for time-consuming bacterial culture while directly detecting pathogen genetic material, reducing detection time from days to hours while maintaining identification accuracy through sequence-specific detection.
Solution Approach 2:
The patent performs nucleic acid extraction and amplification before detection, preparing the target material in advance. The CRISPR/Cas12a system is pre-programmed with guide RNAs specific to pathogen sequences, enabling rapid detection without waiting for colony formation. This preliminary preparation of detection reagents and target nucleic acids accelerates the overall detection process.
2Reliability
If traditional colony culture method is used for pathogen detection, then pathogen identification can be achieved, but contamination may be introduced during the process of bacterial culture and isolation, resulting in false-positive test results
Solution Approach 1:
The patent replaces the open colony culture system with a closed molecular detection system. Nucleic acid extraction and PCR amplification occur in closed tubes, minimizing exposure to environmental contaminants. The CRISPR/Cas12a detection occurs in a closed well plate system, eliminating the contamination risks associated with open plate culture and bacterial manipulation.
Solution Approach 2:
The patent uses nucleic acid as an intermediary target instead of directly handling live bacteria. By detecting pathogen-specific DNA or RNA sequences, the system avoids direct contact with bacterial cultures that could lead to contamination. The nucleic acid target is highly specific and can be detected without culturing, eliminating cross-contamination between samples.
3Adaptability or versatility
If traditional colony culture method is used for pathogen detection, then comprehensive phenotypic characterization can be obtained, but the method is not suitable for anaerobic pathogenic bacteria or other strains hard to be cultured in common conditions
Solution Approach 1:
The patent changes the detection parameter from phenotypic characteristics (which require live bacterial growth under specific conditions) to genotypic characteristics (nucleic acid sequences). This parameter change allows detection of any pathogen that can be genetically identified, including anaerobic bacteria and fastidious organisms that cannot be cultured under standard conditions, thereby expanding detection applicability while maintaining reliability through sequence-specific amplification.
Solution Approach 2:
The patent develops a universal detection platform using PCR and CRISPR/Cas12a that can detect multiple different pathogens through the use of different guide RNAs targeting specific pathogen sequences. This universal system works for aerobic and anaerobic bacteria, Gram-positive and Gram-negative bacteria, and other diverse pathogens, eliminating the need for pathogen-specific culture conditions while maintaining high detection reliability through molecular 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 a high positive detection rate with improved specificity and speed, enabling timely and accurate identification of pathogens, thereby enhancing clinical diagnosis and treatment of severe pneumonia.
Implementation Method 1
detecting presence or absence of a pathogen specific nucleic acid fragment in the sample
Implementation Method 2
amplifying and detecting specific DNA sequences using CRISPR/Cas12a's trans cleavage activity
Implementation Method 3
detecting specific DNA sequences using CRISPR/Cas12a's trans cleavage activity
Data Source
AI summary
Provided are a plurality of pathogen specific nucleic acid fragments. Also provided is a method for identifying one or more pathogens, comprising: 1) providing a sample that may comprise the pathogens; and 2) detecting whether a pathogen specific nucleic acid fragment exits in the sample or not or detecting the content of pathogen specific nucleic acid fragment in the sample, wherein the presence or absence of the pathogen specific nucleic acid fragment or the content thereof respectively reflects the presence or absence of a pathogen corresponding to the pathogen specific nucleic acid fragment in the sample or the content thereof. The provided pathogen specific nucleic acid fragment can be applied to rapid identification of pathogens, and in clinical application, the positive detection rate is high and the detection period is short.