Multiplex Branched DNA Signal Amplification for Citrus Pathogen Detection
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Solution Overview
Problem
Current methods for detecting citrus pathogens are inefficient and lack sensitivity, making it difficult to accurately and quickly identify multiple pathogens in a single sample, particularly in high-throughput screenings.
Innovation Solution
The development of a multiplex branched signal amplification reaction method using specific probes for citrus DNA pathogens, including HLB, citrus greening, Candidatus Liberibacter asiaticus, citrus canker, witches' broom, CVC, and citrus stubborn disease, along with a housekeeping citrus gene as an internal control, allowing for simultaneous detection in a single sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multiple citrus pathogens are detected using conventional methods, then detection accuracy is improved, but detection time and operational complexity increase significantly
Solution Approach 1:
The patent combines multiple pathogen detection assays into a single multiplex reaction system. Different pathogen-specific probes (targeting HLB, canker, witches' broom, CVC, and stubborn disease) are simultaneously detected in one reaction well using distinct fluorescent labels, eliminating the need for separate assays for each pathogen and thereby reducing detection time while maintaining accuracy
Solution Approach 2:
The invention creates a universal detection platform that can identify multiple citrus pathogens using a common reaction system and detection methodology. The multiplex assay framework allows a single test to screen for five different pathogens, making the system multi-functional and highly efficient for routine diagnostics
2Adaptability or versatility
If multiple citrus pathogens are detected using conventional methods, then comprehensive pathogen identification is improved, but procedural complexity increases
Solution Approach 1:
Multiple pathogen detection procedures are merged into a single standardized protocol. The multiplex assay uses a unified reaction mixture containing all necessary probes and reagents, which are incubated together in one well, simplifying the workflow compared to performing separate assays for each pathogen
Solution Approach 2:
The invention establishes a universal detection platform that handles multiple pathogens through a single procedural framework. The system uses standardized reagents, incubation conditions, and detection methods that apply to all target pathogens, reducing the need for pathogen-specific procedural variations
3Measurement precision
If sensitivity of pathogen detection is improved, then detection of low-pathogen loads is enhanced, but false positive rates may increase
Solution Approach 1:
The patent employs branched DNA (bDNA) signal amplification technology as an intermediary mechanism. The bDNA probes hybridize to target pathogen sequences and amplify the fluorescent signal through their branched structure, enabling detection of low-pathogen loads while maintaining specificity through sequence-complementary hybridization, thus reducing false positives
Solution Approach 2:
The invention optimizes hybridization conditions and signal detection parameters to enhance sensitivity. By carefully controlling temperature, salt concentration, and probe concentrations, the assay achieves high sensitivity for detecting low-pathogen loads while maintaining stringency to prevent non-specific binding and false positives
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 method provides an accurate, efficient, and quick means of detecting multiple citrus pathogens, suitable for high-throughput screenings, and can be used in quarantine and certification programs, ensuring effective disease management.
Implementation Method 1
a multiplex branched signal amplification reaction
Data Source
AI summary
The present invention provides methods and compositions for detecting multiple citrus pathogens using a multiplex branched DNA signal amplification reaction.