Multiplex qPCR Assay for cfDNA Integrity Without DNA Purification
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Solution Overview
Problem
Current cancer diagnosis and monitoring methods are invasive, expensive, and suffer from inaccuracies due to well-to-well variability, lack of an internal PCR control, and inefficiencies in single-plex amplification, particularly in assessing cell-free DNA (cfDNA) integrity and concentration in biological fluids.
Innovation Solution
A multiplexed quantitative polymerase chain reaction (qPCR) method that simultaneously assays retrotransposon interspersed element markers, including an internal positive control, to accurately quantify cfDNA integrity and concentration in blood plasma or serum, using independent short and long nucleic acid fragments of retrotransposable elements like ALU and SVA, without prior DNA purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If single-plex amplification is used to assay cfDNA, then the assay can be performed with simple equipment and protocols, but well-to-well variability increases and measurement precision deteriorates
Solution Approach 1:
The patent combines multiple amplification reactions into a single multiplex qPCR reaction. Specifically, it simultaneously amplifies short cfDNA fragments (indicating degradation), long cfDNA fragments (indicating integrity), and an internal positive control target in one reaction mixture. This merging eliminates well-to-well variability by normalizing all measurements to the same reaction conditions, thereby improving measurement precision while maintaining manageable assay complexity.
Solution Approach 2:
The multiplex qPCR assay performs multiple functions simultaneously: it quantifies cfDNA integrity (via long fragment amplification), assesses cfDNA degradation (via short fragment amplification), and monitors PCR inhibition (via internal positive control). This multi-functionality is achieved within a single reaction system using universally applicable qPCR technology, resolving the contradiction between assay simplicity and measurement precision.
2Device complexity
If no internal PCR control is included to reduce assay complexity, then the device complexity decreases, but reliability deteriorates due to inability to monitor PCR inhibitors
Solution Approach 1:
The assay includes an internal positive control that serves itself by automatically monitoring for PCR inhibitors within the reaction mixture. The control target is amplified alongside the cfDNA targets, and its amplification efficiency provides real-time feedback on reaction quality. This self-monitoring mechanism ensures reliability without requiring separate control reactions, thus not significantly increasing overall assay complexity.
3Measurement precision
If DNA purification is performed before qPCR to improve measurement precision, then cfDNA quantification accuracy improves, but loss of substance increases and productivity decreases
Solution Approach 1:
The patent uses an internal positive control as an intermediary to bridge the gap between direct qPCR on crude samples and purified DNA samples. The control target, which is added in known quantities, serves as a reference that allows accurate quantification of cfDNA targets even in the presence of contaminants. This intermediary approach enables precise measurement without requiring DNA purification, thereby preventing cfDNA loss and maintaining high productivity.
4Measurement precision
If multiple separate qPCR reactions are performed to assess cfDNA integrity and concentration, then measurement precision improves, but loss of time increases and productivity decreases
Solution Approach 1:
The patent merges multiple separate qPCR assessments into a single multiplex reaction. It simultaneously measures short cfDNA fragments (for degradation assessment), long cfDNA fragments (for integrity assessment), and internal positive control amplification. By combining these measurements in one reaction tube with one amplification cycle, the assay achieves comprehensive cfDNA characterization without the time loss associated with multiple sequential reactions.
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 provides a rapid, affordable, and high-throughput clinical test for cancer diagnosis, prognosis, and surveillance by enhancing accuracy and reducing labor and costs, while overcoming variability and incorporating an internal control for reaction validation.
Implementation Method 1
using a quantitative polymerase chain reaction (qPCR) method to separately and simultaneously quantitate the short nucleic acid fragment and the long nucleic acid fragment
Data Source
AI summary
A retrotransposable element based multiplexed qPCR assay to robustly quantitate and distinguish cell free DNA integrity and concentration in blood plasma and serum is described. The multiplexed system for characterizing cancer in humans includes a sample of serum, plasma, urine, or other biological fluid, the sample comprising cell free DNA, the cell free DNA comprising long and short retrotransposable element targets and an added internal positive control, the long and short targets being independent of each other, a distinctly labeled TaqMan probe corresponding to each target, a forward primer and a reverse primer corresponding to each target, a DNA standard for generating standard curves, a qPCR system for amplifying the targets and a qPCR data analysis system. The assay provides an accurate, minimally-invasive, rapid, high-throughput, and cost-effective method with the potential to complement or replace existing methods for detection, diagnosis, prognosis, treatment monitoring and/or surveillance of cancer, thereby improving patient outcomes.