dqPCR Method for Wide Concentration Range Nucleic Acid Quantification
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Solution Overview
Problem
Current digital PCR methods face limitations in detecting nucleic acid targets with wide concentration ranges, particularly in clinical samples, as they require sample dilution, which can lead to reduced sensitivity and increased operational complexity and cost due to the need for more reaction wells.
Innovation Solution
A digital and quantitative PCR method that simultaneously performs real-time quantitative polymerase chain reaction (qPCR) and digital PCR, allowing for the direct quantification of low-concentration targets and conversion of high-concentration targets' Cq values to copy numbers, expanding the dynamic range without sample dilution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If digital PCR is used to measure sample concentration, then absolute quantification can be achieved without calibration curves, but the dynamic range is limited and sample concentration must be kept low
Solution Approach 1:
The patent segments the detection process into two distinct modes: digital PCR mode for absolute quantification of low-concentration targets and qPCR mode for high-concentration targets. This segmentation allows each mode to operate in its optimal concentration range, thereby resolving the contradiction between measurement precision and dynamic range adaptability
Solution Approach 2:
The patent implements a dual-mode PCR system that can universally handle both digital PCR and qPCR functions within a single platform. This multi-functionality enables the system to adapt to different concentration ranges without requiring separate measurement systems, thus expanding the dynamic range while maintaining absolute quantification capabilities
2Adaptability or versatility
If sample dilution is performed to enable digital PCR measurement of high-concentration targets, then the concentration falls within the applicable range, but sensitivity is reduced due to over-dilution of low-concentration targets
Solution Approach 1:
The patent introduces dynamic mode selection capability that allows the system to adaptively switch between digital PCR mode and qPCR mode based on the concentration of the target nucleic acid. This dynamics enables high-concentration targets to be measured in qPCR mode without dilution while low-concentration targets are measured in digital PCR mode, thereby maintaining detection sensitivity across the full concentration range
3Reliability
If the number of reaction wells is increased to meet digital PCR requirements for high-concentration samples, then enough negative reaction wells are available, but the difficulty, cost and time of detection increase
Solution Approach 1:
The patent changes the operational parameters of the PCR system by introducing a dual-mode capability that can operate in either digital PCR mode or qPCR mode. This parameter change allows the system to maintain reliable measurement of high-concentration targets in qPCR mode without requiring an increased number of reaction wells, thereby reducing device complexity while preserving measurement reliability
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 effectively detects both high- and low-concentration nucleic acid targets in a single run, enhancing the dynamic range and operational convenience by eliminating the need for sample dilution and reducing sensitivity issues, while maintaining high accuracy and linearity.
Implementation Method 1
perform a digital and quantitative real-time PCR on a nucleic acid sample
Implementation Method 2
a Cq value of the high-concentration nucleic acid target is detected
Data Source
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AI summary
A digital and quantitative PCR (dqPCR) measuring method for a nucleic acid sample is provided, including providing a testing plate having a plurality of reaction wells, so as to perform a dqPCR reaction on the nucleic acid sample. Especially for the nucleic acid sample to be tested having a plurality of nucleic acid targets with different concentration ranges of wide variations, digital PCR and quantitative PCR can be performed simultaneously, so as to quantify the copy number of nucleic acid targets.