Internal Standard Oligonucleotides for Accurate cfDNA qPCR Quantification
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Solution Overview
Problem
Current methods for quantifying circulating cell-free DNA (cfDNA) in bodily fluids suffer from inaccuracies due to DNA loss during extraction, DNA degradation, and variations in detection and quantification methods, leading to unreliable results, especially in the context of diseases like COVID-19 caused by SARS-COV-2 variants.
Innovation Solution
The use of internal standard oligonucleotides and primers for multiplex quantitative real-time PCR, including a double-stranded oligonucleotide sequence similar to human β-actin gene, allows for accurate detection and quantification of cfDNA by incorporating an internal standard oligonucleotide that stabilizes the quantification process and enhances sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If external standard and PicoGreen labels are used for quantification, then the method is simple to operate, but the measurement precision and reliability are low due to DNA loss during extraction and degradation
Solution Approach 1:
The patent introduces an internal standard oligonucleotide as an intermediary substance that is added to the test sample before extraction. This internal standard serves as a reference that experiences the same extraction and degradation processes as the target cfDNA, allowing for correction of losses and variations. The internal standard enables accurate quantification by comparing the recovery of the known amount of internal standard against the target analyte, thereby resolving the contradiction between operational simplicity and measurement precision.
2Loss of time
If cfDNA is detected directly without amplification using PicoGreen fluorophores, then the method is quick to perform, but the sensitivity is low and variation between tests is significant
Solution Approach 1:
The patent applies preliminary action by adding the internal standard oligonucleotide to the test sample before the extraction and detection processes. This allows the internal standard to undergo the same preparation steps as the target cfDNA, establishing a reference point early in the workflow. The internal standard is then amplified together with the target analyte in the subsequent qPCR step, enabling sensitive detection while maintaining a streamlined process that does not significantly increase time consumption.
3Measurement precision
If quantitative analysis is performed using gene amplification means, then the sensitivity is improved, but the manufacturing precision is reduced due to nucleic acid loss during extraction and DNA degradation
Solution Approach 1:
The patent implements feedback by using the internal standard oligonucleotide to monitor and correct for extraction efficiency and degradation throughout the process. The internal standard undergoes the same extraction, purification, and amplification steps as the target cfDNA, and its recovery rate provides feedback information about the efficiency of each step. This feedback mechanism allows for calculation of correction factors that compensate for losses and variations, thereby maintaining both sensitivity and manufacturing precision.
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 approach provides stable and accurate quantification of cfDNA, enabling precise assessment of disease severity, particularly in SARS-COV-2 infections, with high sensitivity and specificity in predicting infection outcomes.
Implementation Method 1
multiplex quantitative real-time PCR
Implementation Method 2
primers and probes for multiplex quantitative real-time PCR
Data Source
AI summary
Provided herein are internal standard oligonucleotides, primers, probes and kits for the detection and quantification of cell-free DNA using multiplex quantitative real-time PCR.