Isothermal Nucleic Acid Quantification via Digital Segmentation
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Solution Overview
Problem
Current nucleic acid detection and quantification methods, such as real-time PCR and LAMP, face challenges with accuracy and complexity, particularly in requiring precise liquid adjustments and multiple container handling, which complicates the process and reduces sensitivity.
Innovation Solution
A nucleic acid detection method utilizing a substrate with multiple detection regions, where a reaction field is formed with a sample, primer set, and amplification enzyme, maintained under isothermal conditions, allowing for accurate detection and quantification based on the number of positive detection regions and rise time of detection signals without the need for liquid dilution or multiple container handling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If digital PCR method is used to achieve high accuracy in quantification or detection, then measurement precision is improved, but device complexity increases due to requiring adjustment of reaction liquid concentration and dispensing into many containers
Solution Approach 1:
The substrate is divided into multiple detection regions (e.g., 96, 384, or 1536 regions) that can simultaneously perform digital PCR reactions. This segmentation allows parallel processing of many samples in a single container, eliminating the need for manual dispensing into multiple containers while maintaining the quantification accuracy of digital PCR method.
Solution Approach 2:
The invention creates multiple copies of detection regions on a single substrate, each capable of independent detection. This allows the system to perform what would traditionally require many separate containers using only one container with multiple detection zones, thereby simplifying operations while preserving measurement precision.
2Reliability
If real-time PCR or LAMP method is used for nucleic acid detection, then sensitivity is improved through amplification, but manufacturing precision deteriorates due to low accuracy in analysis
Solution Approach 1:
By dividing the reaction system into multiple discrete detection regions on a single substrate, the invention enables digital PCR analysis where each region independently detects and quantifies nucleic acid molecules. This segmentation provides absolute quantification capability that overcomes the relative quantification limitations of real-time PCR and LAMP, thereby improving analysis accuracy while maintaining high detection sensitivity through amplification.
Solution Approach 2:
The invention replaces the mechanical/chemical detection systems of real-time PCR (fluorescence curves) and LAMP (visual changes) with electrical detection systems that measure current changes in each detection region. This substitution enables more precise and automated quantification, improving manufacturing precision while maintaining the sensitivity benefits of amplification methods.
3Measurement precision
If digital PCR method is used to achieve high accuracy, then measurement precision is improved, but ease of operation deteriorates due to complicated liquid adjustment and multiple container handling
Solution Approach 1:
The invention merges multiple detection regions onto a single substrate that fits in one container. This allows the entire digital PCR assay to be performed in a single container without the need for adjusting liquid concentrations or handling multiple containers, dramatically improving ease of operation while maintaining the high measurement precision of digital PCR method.
Solution Approach 2:
The substrate with multiple detection regions serves multiple functions simultaneously: it provides reaction chambers for amplification, detection zones for signal measurement, and quantification units for data analysis. This multi-functionality eliminates the need for separate steps of liquid adjustment and multiple container handling, making the high-precision digital PCR method as easy to operate as conventional methods.
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 enhances sensitivity and accuracy, enabling the detection of 1 to 10^9 copies/mL of target nucleic acid with improved precision and simplicity, reducing operational complexity and increasing reliability across various samples.
Implementation Method 1
a primer set for isothermally amplifying the first sequence to obtain an amplification product, and an amplification enzyme
Data Source
AI summary
According to one embodiment, a method of quantifying a target nucleic acid containing a first sequence in a sample is provided. The method includes preparing a substrate on which a plurality of detection regions are arranged, forming a reaction field by placing, on the substrate, a reaction liquid containing a sample, a primer set, and an amplification enzyme, maintaining the reaction field in an isothermal amplification condition, detecting a detection signal for each of the detection regions, determining, for each of the plurality of detection regions, whether positive or negative and detecting or quantifying the target nucleic acid based on the number of positive and/or a rise time of each of the positive detection signal.