Real-Time Digital PCR Partition Analysis for Wide Concentration Range
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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 reaction images in real-time, allowing for partition classification and concentration calculation through Poisson probability distribution and cycle threshold values.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If end-point method is used to measure fluorescence intensity after PCR completion, then the presence or absence of target nucleic acids can be confirmed, but measurement precision deteriorates in low-concentration samples due to inability to distinguish intermediate fluorescence intensities
Solution Approach 1:
The patent performs real-time fluorescence measurement during the PCR amplification process rather than only at the endpoint. By capturing fluorescence intensity at multiple time points throughout the PCR cycles, the system can track the amplification curve and identify the cycle threshold (Ct) value, which provides precise quantification information before the reaction reaches completion. This preliminary action during the process enables accurate measurement of low-concentration samples.
2Measurement precision
If Poisson probability distribution is applied to calculate target nucleic acid numbers, then quantification can be performed, but false positives and false negatives occur when fluorescence intensity is intermediate between positive and negative partitions
Solution Approach 1:
The patent uses real-time fluorescence measurement to generate amplification curves that provide feedback on the amplification progress of each partition. By analyzing the shape and characteristics of these curves, the system can distinguish between true positive amplifications and background noise more reliably than endpoint measurement alone. The real-time data allows for setting dynamic thresholds and applying quality control criteria to reduce false positives and false negatives.
3Measurement precision
If serial dilution and retesting is performed to resolve all-positive results, then target nucleic acid numbers can be calculated, but measurement time and process complexity increase
Solution Approach 1:
The patent performs real-time fluorescence measurement continuously throughout the entire PCR amplification process in all partitions simultaneously. This continuous monitoring allows the system to detect the amplification signal in high-concentration samples during the PCR cycles themselves, eliminating the need for post-PCR serial dilution and retesting. The useful action of measurement continues uninterrupted from the beginning to the end of PCR, providing quantification information for all concentration ranges in a single test.
4Measurement precision
If droplet digital PCR method is used with special droplet generator and multiple equipment, then partitioning into droplets can be achieved, but device complexity and operational difficulty increase
Solution Approach 1:
The patent combines the partitioning function and the PCR amplification function into a single integrated microfluidic chip system. The microfluidic chip contains pre-formed partitions (wells or chambers) that can directly receive and amplify the sample without requiring separate droplet generation equipment. This merging of functions eliminates the need for multiple separate devices (droplet generator, transfer equipment, etc.) and simplifies the overall system while maintaining the partitioning capability essential for digital PCR.
5Measurement precision
If number of partitions is increased to improve limit of detection, then sensitivity for low-concentration samples improves, but false positives and false negatives increase due to more intermediate intensity partitions
Solution Approach 1:
The patent performs real-time fluorescence measurement during the PCR amplification process to generate amplification curves for each partition. By analyzing the dynamics of fluorescence increase throughout the PCR cycles, the system can distinguish between true positive amplifications (which show characteristic exponential growth) and background noise or intermediate signals (which do not show proper amplification kinetics). This preliminary action during the process provides kinetic information that improves classification accuracy even with a large number of partitions.
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
Enables accurate real-time analysis of high and low-concentration samples, reducing false errors and expanding the measurable dynamic range, achieving precise quantification without the need for additional equipment or skilled personnel.
Implementation Method 1
a CMOS photosensor array located on a bottom surface of the well array and configured to capture a reaction image of the analysis target sample filled in a plurality of partitions provided in the well array in real-time
Data Source
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.