Multiplex Nucleic Acid Detection via Temperature-Dependent Signal Differentiation
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Solution Overview
Problem
Conventional real-time PCR methods struggle to differentiate and quantify multiple target nucleic acid sequences using a single type of label in a single reaction vessel, leading to inefficiencies and inaccuracies due to overlapping signal detection and limited temperature range adjustments.
Innovation Solution
A method utilizing signal-change values calculated at specific temperature intervals for duplexes with labeled oligonucleotides, allowing for the detection of multiple target nucleic acid sequences by differentiating signal changes between narrow temperature ranges, thereby eliminating interference and improving accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If different types of labels are used to detect multiple target nucleic acid sequences, then detection accuracy is improved, but device complexity and cost increase
Solution Approach 1:
The patent applies parameter changes by utilizing different detection temperatures to differentiate between multiple target nucleic acid sequences. Each target sequence is detected at a specific detection temperature where its corresponding amplicon exhibits maximum signal intensity, allowing multiple sequences to be detected using a single label type without increasing device complexity
Solution Approach 2:
The patent introduces temperature as an additional dimension for differentiation. Instead of using different labels (one-dimensional differentiation), the method employs temperature-dependent signal detection, where each target sequence is detected at its optimal detection temperature, effectively adding a temporal/thermal dimension to the detection process
2Adaptability or versatility
If melting analysis is used to detect multiple target nucleic acid sequences, then detection capability is improved, but analysis time increases significantly
Solution Approach 1:
The patent performs preliminary action by pre-determining optimal detection temperatures for each target nucleic acid sequence based on their respective amplicon Tm values. During the real-time PCR process, signal detection is performed at these predetermined temperatures at each amplification cycle, eliminating the need for time-consuming post-amplification melting curve analysis
Solution Approach 2:
The patent substitutes the mechanical melting analysis process with a temperature-dependent signal detection system. Instead of analyzing melting curves after amplification, the method detects signals at specific temperatures during the amplification process itself, replacing a time-intensive analytical step with an integrated detection approach
3Measurement precision
If detection temperatures are narrowly separated for multiple targets, then detection precision is improved, but signal differentiation becomes difficult
Solution Approach 1:
The patent applies local quality by optimizing detection conditions for each specific target sequence. Each target nucleic acid sequence has its own optimal detection temperature determined by its amplicon Tm value, allowing precise detection even when temperature intervals are narrow. The signal intensity at each detection temperature reflects the specific target present, enabling differentiation without requiring large temperature separations
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 enables reliable and accurate detection of multiple target nucleic acid sequences with reduced risk of false positives, using a single type of label and narrower temperature intervals, enhancing the precision and efficiency of the detection process.
Implementation Method 1
signal-generating compositions for generating a fluorescent signal being detectable in a proportional manner with the amount of the target molecule
Implementation Method 2
labeled probes or primers specifically hybridized with target nucleic acid sequences
Implementation Method 3
The melting analysis using Tm difference permits to detect a plurality of target nucleic acid sequences
Data Source
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AI summary
The method of the present invention enables efficient detection of a plurality of target nucleic acid sequences in one detection channel, by obtaining a data set of cycle/signal-change value.