Multiplexed qPCR Assay for cfDNA Integrity Measurement
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Solution Overview
Problem
Current methods for assessing circulating cell-free DNA (cfDNA) integrity and concentration in cancer diagnosis and monitoring are invasive, costly, and lack sensitivity, particularly for early-stage colorectal cancer, and existing assays are limited by well-to-well variability and poor primer specificity, preventing accurate multiplexed analysis.
Innovation Solution
Development of a multiplexed quantitative polymerase chain reaction (qPCR) assay using retrotransposable element (RE) targets, allowing for simultaneous measurement of cfDNA integrity and concentration in a single reaction, with the inclusion of internal PCR controls to enhance accuracy and specificity, enabling rapid, cost-effective, and minimally invasive cancer monitoring.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If single-plex PCR is used to amplify each target sequence separately, then primer specificity is improved, but well-to-well variability increases and measurement precision deteriorates
Solution Approach 1:
The patent combines multiple target sequence amplifications into a single multiplexed PCR reaction well, allowing simultaneous amplification of multiple ALU element targets (e.g., 247 bp and 115 bp fragments) in one reaction. This merging approach eliminates well-to-well variability by removing the need for separate reactions, while maintaining primer specificity through carefully designed primer sets that specifically bind to their intended targets within the multiplexed system.
2Reliability
If single-plex amplification is used for each target, then primer specificity is improved, but the assay becomes more labor-intensive and less cost-effective
Solution Approach 1:
The patent merges multiple single-plex amplification steps into a single multiplexed PCR reaction, thereby reducing the number of separate reactions, reagent preparations, and data analysis steps required. This increases productivity and cost-effectiveness while maintaining reliability through optimized primer design that ensures specific amplification of multiple targets simultaneously.
Solution Approach 2:
The multiplexed PCR assay serves multiple functions within a single reaction system: it simultaneously quantifies different cfDNA fragments (247 bp and 115 bp), assesses cfDNA integrity, and can include internal controls all in one well. This multi-functionality improves productivity without sacrificing the specificity needed for accurate measurement.
3Device complexity
If conventional single-plex PCR is used, then internal PCR control cannot be incorporated, but the assay setup is simpler
Solution Approach 1:
The patent incorporates an internal PCR control into the multiplexed reaction system, combining the target amplification and control amplification in the same well. This allows simultaneous monitoring of reaction success and target quantification, improving reliability by confirming that PCR inhibitors are not present and that the reaction conditions are appropriate, while the overall setup remains relatively simple.
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 multiplexed qPCR assay provides highly sensitive and specific detection of cancer, capable of distinguishing between normal and cancerous cfDNA, facilitating early diagnosis and monitoring of cancer progression with improved patient outcomes and reduced exposure to radiation.
Implementation Method 1
multiplexed quantitative polymerase chain reaction (qPCR) assay
Data Source
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AI summary
A retrotransposable element-based multiplexed quantitative polymerase chain reaction (qPCR) assay system to quantitate and distinguish cell free DNA integrity and concentration in blood, plasma, and serum as a measure of minimum residual disease, therapeutic effectiveness, neoadjuvant effectiveness in a patient having stage I, stage II, stage III, or stage IV cancer, and disease progression, thereby improving patient outcomes.