Digital Real-Time PCR With CMOS Imaging for Wide-Range Quantification
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Solution Overview
Problem
Existing digital PCR technologies face challenges in accurately distinguishing between positive and negative partitions, particularly in low-concentration samples, leading to false positives and negatives, and have limited dynamic range for quantification, making it difficult to measure samples beyond existing concentration limits.
Innovation Solution
A digital real-time PCR method using a cartridge with a microfluidic chamber, well array, and CMOS photosensor array to capture real-time reaction images, enabling partition classification and concentration calculation through Poisson probability distribution and cycle threshold (Ct) values.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If end-point method is used to determine positive/negative partitions, then measurement process is simple, but measurement precision deteriorates due to inability to distinguish intermediate intensity partitions
Solution Approach 1:
The patent performs real-time fluorescence intensity monitoring during the PCR amplification process before the end-point is reached. By capturing fluorescence data at multiple time points throughout the amplification cycles, the system can identify positive partitions based on their amplification curves rather than relying solely on final end-point intensity, thereby improving classification accuracy while maintaining operational simplicity.
Solution Approach 2:
The system uses real-time fluorescence intensity feedback to dynamically adjust partition classification. By continuously monitoring fluorescence signals during PCR amplification and comparing them against threshold values or amplification patterns, the system can accurately distinguish positive, negative, and intermediate partitions, resolving the precision issue while keeping the process straightforward.
2Measurement precision
If number of partitions is increased to improve LOD of low-concentration samples, then limit of detection improves, but reliability deteriorates due to increased intermediate intensity partitions
Solution Approach 1:
The patent employs dynamic thresholding and real-time amplification curve analysis instead of static end-point classification. By evaluating fluorescence intensity trends and amplification kinetics throughout the PCR process, the system can reliably classify partitions even when using a large number of partitions, thereby maintaining both low detection limits and high quantitative accuracy.
Solution Approach 2:
The system changes the parameter used for classification from static end-point fluorescence intensity to dynamic amplification curve characteristics. By analyzing the shape, slope, and progression of fluorescence curves over multiple cycles, the system can distinguish true positive signals from intermediate or noise signals, maintaining reliability while improving detection sensitivity through increased partition numbers.
3Measurement precision
If dropletization method is used for partitioning, then measurement precision improves for digital PCR, but device complexity increases due to requirement of droplet generator and multiple equipment
Solution Approach 1:
The patent merges the partitioning function with the PCR reaction vessel itself by using a microplate or well-based system where each well serves as a partition. This eliminates the need for separate droplet generation equipment while maintaining the ability to perform digital PCR quantification, thereby reducing device complexity while preserving measurement precision.
Solution Approach 2:
The invention extracts the droplet generation step from the overall system by using pre-formed discrete partitions (wells or microcompartments) in a microplate. This removes the complex droplet generator apparatus while retaining the essential partitioning function needed for digital PCR, simplifying the device architecture without compromising quantification accuracy.
4Reliability
If real-time fluorescence monitoring is implemented, then reliability improves by enabling accurate partition classification, but use of energy increases due to continuous imaging
Solution Approach 1:
The patent implements periodic fluorescence monitoring at strategically selected time points during PCR amplification rather than continuous monitoring. By capturing images at key cycles where amplification divergence between positive and negative partitions becomes apparent, the system achieves reliable classification with reduced energy consumption compared to uninterrupted real-time imaging.
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 provides accurate real-time analysis for high and low-concentration samples, reducing false positives and negatives, and allows for precise quantification across a wide concentration range without the need for additional equipment or skilled personnel.
Implementation Method 1
a raw data acquisition step (S20) of acquiring raw data by capturing the reaction image of the analysis target sample filled in the plurality partitions in real time through the CMOS photosensor array
Implementation Method 2
Polymerase chain reaction (PCR) is a representative gene amplification technology and consists of three steps of DNA denaturation, primer annealing, and DNA extension, and since each step depends on the temperature of the sample, DNA may be amplified by repeatedly changing the temperature of the sample
Data Source
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AI summary
The present invention relates to a digital real-time PCR analysis method, and the purpose of the present invention is to provide a digital real-time PCR analysis method capable of digital real-time PCR analysis of a low-concentration or a high-concentration sample which is beyond an existing measurement limit, i.e., samples of a wide concentration range.