Dual-Probe qPCR Method for Simultaneous Mutation Detection
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Solution Overview
Problem
The existing fluorescence quantitative PCR methods require multiple primer and probe screenings for each mutation detection, consuming samples and prolonging the testing cycle, leading to increased costs and variability in results due to reference gene expression changes.
Innovation Solution
A dual-probe method for fluorescence quantitative PCR using a Taqman dual-probe system with shared primers, where the detection probe targets a hotspot mutation site and the reference probe targets an adjacent sequence, both labeled with different fluorescent markers, allowing for simultaneous detection of multiple adjacent mutations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multiple primer and probe screenings are performed for each mutation detection, then detection accuracy is improved, but sample consumption increases and testing time is prolonged
Solution Approach 1:
The patent combines multiple detection functions into a single qPCR reaction system. By designing a dual-probe system where one probe detects the reference gene and another probe detects the mutation-specific sequence, multiple screenings are merged into one simultaneous detection process, thereby reducing testing time and sample consumption while maintaining detection accuracy
Solution Approach 2:
The invention creates a universal detection platform that can screen for multiple different mutations using the same qPCR system and reagents. The dual-probe design allows the system to function as both a reference gene detector and a mutation-specific detector, eliminating the need for separate screenings for each mutation type
2Measurement precision
If multiple primer and probe screenings are performed for each mutation detection, then detection accuracy is improved, but testing cost increases
Solution Approach 1:
The patent merges multiple detection functions into a single qPCR reaction system. By designing a dual-probe system where one probe detects the reference gene and another probe detects the mutation-specific sequence, multiple screenings are merged into one simultaneous detection process, thereby reducing testing time and sample consumption while maintaining detection accuracy
Solution Approach 2:
The invention creates a universal detection platform that can screen for multiple different mutations using the same qPCR system and reagents. The dual-probe design allows the system to function as both a reference gene detector and a mutation-specific detector, eliminating the need for separate screenings for each mutation type
3Productivity
If reference gene expression levels are used for result interpretation, then quantitative analysis is enabled, but result reliability deteriorates due to expression variability
Solution Approach 1:
The patent introduces a mutation-specific detection probe as an intermediary that directly detects the presence and quantity of mutant sequences. This intermediary approach bypasses the need for complex reference gene-based normalization, as the detection probe specifically binds to the mutation sequence and provides direct quantitative information about the mutant allele, thereby improving result reliability
Solution Approach 2:
The invention uses fluorescently labeled probes that emit different signals based on binding to target sequences. The detection probe emits fluorescence specifically when bound to the mutation sequence, providing a direct visual and measurable indicator of mutation presence and quantity, eliminating the need for indirect reference gene-based interpretation
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 reduces sample consumption and testing time while enhancing result accuracy and reliability by using a single reaction, enabling effective detection of gene mutations with improved credibility.
Implementation Method 1
According to the principle of fluorescence resonance energy transfer, the emitted fluorescence of the fluorescent reporting group is quenched because it is in close proximity to the quencher group in the intact probe. When the probe is degraded, the fluorescent reporter group and the quencher are separated, and the fluorescence is emitted.
Data Source
AI summary
A dual-probe method for fluorescence quantitative PCR (Polymerase Chain Reaction) is disclosed. The method involves the use of a Taqman dual-probe detection system with the same pair of primers. The dual probes includes a detection probe and a reference probe. The detection probe targets the wild-type sequence at the hotspot mutation site, which can cover multiple adjacent mutations. The reference probe targets the wild-type sequence adjacent to the target sequence. Both probes share the same pair of upstream and downstream primers. This method uses a single reaction qPCR (quantitative PCR) method, which can simultaneously detect multiple adjacent mutations. It effectively saves tissue samples and greatly shortens the testing period. Moreover, the close proximity of the target and reference sequences enhances the reliability and accuracy of the results, making it widely applicable in various fields.