Multiplex qRT-PCR Primers for Orchid Virus Detection
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Solution Overview
Problem
Current methods for detecting Cymbidium mosaic virus (CymMV), Odontoglossum ringspot virus (ORSV), and Cymbidium ringspot virus (CymRSV) in orchids are inefficient, time-consuming, and prone to false positives, with limited multiplex simultaneous detection technology available, necessitating the development of specific primers and probes for rapid, sensitive, and specific detection.
Innovation Solution
Designing specific primers and probes for real-time fluorescence quantitative PCR, including forward and reverse primers and probes for each virus, optimized for simultaneous detection, using conserved sequences of the coat protein genes, and establishing a real-time fluorescence quantitative PCR amplification system with optimized reaction conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR methods are used for virus detection, then qualitative detection can be achieved, but false positives occur due to non-specific amplification and only single virus detection is possible
Solution Approach 1:
The patent combines multiple detection functions into a single multiplex qRT-PCR system that can simultaneously detect CymMV, ORSV, and CymRSV viruses in one reaction, eliminating the need for separate PCR experiments for each virus and reducing false positives through standardized real-time quantification
Solution Approach 2:
The patent introduces fluorescent probes as intermediaries that specifically bind to target virus sequences and emit fluorescence signals, enabling precise quantitative detection without non-specific amplification that causes false positives in conventional PCR
2Ease of operation
If indicator plant method is used for virus detection, then simple operation is achieved, but detection time is long (10-90 days) and sensitivity is low
Solution Approach 1:
The patent replaces the mechanical biological indicator plant method with a molecular biology-based qRT-PCR system that uses chemical reactions and fluorescent detection, reducing detection time from weeks to hours while maintaining ease of operation through standardized protocols
3Reliability
If ELISA method is used for virus detection, then detection can be performed, but sensitivity is limited to nanogram level and cannot detect lower concentrations
Solution Approach 1:
The patent changes the detection parameter from nanogram-level protein detection (ELISA) to femtogram-level nucleic acid detection (qRT-PCR), achieving sensitivity improvement by several orders of magnitude through exponential amplification of viral RNA templates
4Adaptability or versatility
If gene chip technology is used for pathogen detection, then comprehensive detection is possible, but the system is complex, requires high skill, and production cost is expensive
Solution Approach 1:
The patent extracts the essential detection function from complex gene chip technology, creating a simplified multiplex qRT-PCR system that uses standard laboratory equipment and straightforward protocols while maintaining the capability to detect multiple viruses simultaneously
5Measurement precision
If uniplex qRT-PCR is used for virus detection, then high sensitivity and specificity are achieved, but detection time and cost are high for multiple viruses
Solution Approach 1:
The patent merges multiple uniplex qRT-PCR reactions into a single multiplex reaction by combining primers and probes for different viruses in one tube, maintaining high sensitivity and specificity while reducing detection time and cost by one-third compared to sequential uniplex testing
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 enables rapid, sensitive, and specific detection of CymMV, ORSV, and CymRSV, reducing detection time and cost by one-third, achieving high sensitivity with the ability to detect as few as 1-10 copies, and ensuring accurate quantification and qualitative analysis in a single test.
Implementation Method 1
real-time fluorescence quantitative PCR method utilizes changes in fluorescence signals to detect changes in the amount of amplification product
Implementation Method 2
real-time quantitative PCR (qRT-PCR), which utilizes changes in fluorescence signals to detect changes in the amount of amplification product in each cycle of PCR amplification reaction
Data Source
AI summary
A set of primers and probes for simultaneous detection of Cymbidium mosaic virus (CymMV), Odontoglossum ringspot virus (ORSV), and Cymbidium ringspot virus (CymRSV) and a method for detecting CymMV, ORSV, and CymRSV, along with a method for their detection, are disclosed. The method involves designing multiplex real-time quantitative PCR detection primers and probes for CymMV, ORSV, and CymRSV and applying these primers and probes to the real-time quantitative PCR simultaneous detection of CymMV, ORSV, and CymRSV. It allows for faster detection of CymMV, ORSV, and CymRSV, taking only one-third of the time compared to uniplex real-time quantitative PCR technology, thereby reducing testing costs by approximately ⅓ to ½ for each sample. The primers and probes are highly specific and sensitive, with a sensitivity as low as 1 to 10 copies. It provides an efficient and feasible detection method for early detection and prevention of CymMV, ORSV, and CymRSV.