Random Emulsification Digital Quantitative Analysis Method
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing digital PCR methods face limitations in achieving absolute quantitative analysis of nucleic acid samples across all concentrations due to the need for equal probability distribution of sample molecules in uniformly sized zones, which restricts sensitivity and accuracy at higher concentrations and requires gradient dilution, increasing operational complexity and cost.
Innovation Solution
A random emulsification digital absolute quantitative analysis method that generates isolated reaction zones or droplets with varying sizes and volumes, allowing for amplification and image analysis to determine the presence of target molecules, enabling accurate calculation of total target molecules in a sample regardless of concentration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If uniformly sized zones are used for digital PCR analysis, then equal probability distribution of sample molecules is achieved, but the dynamic range is severely limited and gradient dilution is required for high concentration samples
Solution Approach 1:
The patent applies local quality by allowing each reaction zone to have different sizes and volumes rather than enforcing uniformity. This enables zones to be optimized for different concentration ranges, with larger zones capturing more molecules from dilute samples and smaller zones providing resolution for concentrated samples, all within a single assay without requiring gradient dilution.
Solution Approach 2:
The patent introduces dynamics by making the zone sizes variable and non-uniform rather than static and equal. The system dynamically adapts to different sample concentrations by using a distribution of zone sizes, allowing the same system to handle both low and high concentration samples effectively without manual intervention for dilution.
2Measurement precision
If gradient dilution is performed multiple times to test high concentration samples, then ideal response results are obtained, but operational complexity and time cost increase
Solution Approach 1:
The patent applies segmentation by dividing the sample into multiple reaction zones with different sizes simultaneously, rather than requiring sequential dilution steps. Each zone segment is optimized to capture a specific range of molecule concentrations, allowing all concentration ranges to be tested in parallel within a single reaction system.
Solution Approach 2:
The patent implements preliminary action by pre-establishing a distribution of zone sizes before sample addition. This pre-configured heterogeneous zone system is ready to accommodate molecules across the full concentration range from the outset, eliminating the need for preliminary dilution steps and direct-to-quantification analysis.
3Measurement precision
If microfluidic technology is used to form uniformly sized zones, then digital PCR functionality is achieved, but additional technical difficulty and economy cost are incurred
Solution Approach 1:
The patent applies copying by using conventional PCR well formats as templates for the reaction zones, rather than requiring specialized microfluidic devices. The heterogeneous zone sizes are achieved through standard laboratory techniques, copying the simplicity of traditional PCR setups while incorporating the innovative element of variable zone volumes to enable absolute quantification.
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 allows for precise absolute quantitative analysis of nucleic acid samples at any concentration, improving sensitivity and accuracy while reducing operational complexity and cost by eliminating the need for gradient dilution.
Implementation Method 1
performing random emulsification processing on a system to be emulsified in a preset container to obtain several isolated reaction zones or droplets
Implementation Method 2
performing PCR amplification on each reaction zone at the same time, so as to only generate an amplified fluorescence signal (or other signals) in zones containing one or more target DNAs/RNAs
Implementation Method 3
generate an amplified fluorescence signal (or other signals) in zones containing one or more target DNAs/RNAs
Data Source
AI summary
A random emulsification digital absolute quantitative analysis method includes: performing random emulsification processing on a system to be emulsified to obtain several isolated reaction zones or droplets; determining the total number and volume information of the various reaction zones or droplets, the presence of target molecules to be tested in the respective reaction zones or droplets, and the number of reaction zones or droplets which do not contain the target molecules by combining acquired target images comprising image regions corresponding to the amplified reaction zones or droplets, and analyzing the target images; and accurately calculating the volume information of the various reaction zones or droplets, the presence of the target molecules to be tested in the respective reaction zones or droplets, and the number of reaction zones or droplets which do not contain the target molecules, the total number of target molecules in a sample to be tested.