Plasma Separation Efficiency Assessment Using Dual-Length Real-Time PCR
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Solution Overview
Problem
Existing methods for evaluating plasma separation from whole blood are laborious, expensive, and insufficiently accurate, and require large DNA amounts, making them unsuitable for efficient analysis of circulating cell-free DNA.
Innovation Solution
A real-time PCR assay using co-amplification of a short amplicon (70-150 bps) and a long amplicon (350-600 bps) to determine separation efficiency based on the difference in amplification levels, eliminating the need for absolute quantification and standard curves.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional methods (hemocytometer or flow cytometer) are used to evaluate plasma separation efficiency, then separation assessment is possible, but the methods are laborious, expensive, and insufficiently accurate
Solution Approach 1:
The invention changes the measurement parameter from direct cell counting (hemocytometer/flow cytometer) to DNA fragment length analysis via PCR amplification. By amplifying DNA fragments of different sizes (short cfDNA vs. long gDNA) and comparing their amplification efficiencies, the method achieves higher accuracy in assessing plasma separation efficiency while avoiding complex and expensive equipment
Solution Approach 2:
The invention replaces mechanical/physical measurement systems (hemocytometer manual counting, flow cytometer automated counting) with a molecular biology-based PCR amplification system. This substitution eliminates the need for complex optical equipment and manual operations, reducing both device complexity and operational labor while improving measurement precision
2Measurement precision
If gel electrophoresis is used to determine separation efficiency by detecting long versus short DNA fragments, then separation assessment is possible, but large amounts of DNA are required
Solution Approach 1:
The invention performs preliminary PCR amplification of the DNA samples before detection. By pre-amplifying both short cfDNA fragments and long gDNA fragments using specific primer pairs, the method generates sufficient detectable signal from minimal input DNA, eliminating the need for large DNA quantities required by gel electrophoresis while maintaining the ability to distinguish fragment lengths through amplification efficiency differences
3Measurement precision
If DNA quantification and copy number determination are performed to assess plasma separation, then separation efficiency can be evaluated, but the process becomes complex and time-consuming
Solution Approach 1:
The invention extracts and utilizes only the essential information needed for separation assessment: the relative amplification efficiency difference between short and long DNA fragments. By eliminating unnecessary steps such as absolute DNA quantification, standard curve generation, and copy number determination, the method achieves accurate separation efficiency evaluation in a single PCR reaction, significantly reducing time and procedural complexity
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
Provides a simple, cost-effective, and accurate method to assess plasma separation efficiency without requiring DNA quantification, ensuring high-quality analysis of cell-free DNA by distinguishing between plasma and whole blood DNA fragments.
Implementation Method 1
A real-time PCR assay using co-amplification of a short amplicon (70-150 bps) and a long amplicon (350-600 bps) to determine separation efficiency
Data Source
AI summary
Methods and kits for determining the efficiency of plasma separation from whole blood are provided, using real-time PCR amplification of two amplicons, namely, a short amplicon of e.g. 70-150 bps and a long amplicon of e.g. 350-600 bps. The separation efficiency is determined based on the difference in amplification patterns of the two amplicons.