Surrogate Nucleic Acid Detection for Reproducible qPCR
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Solution Overview
Problem
Current nucleic acid detection methods, such as qPCR, face challenges including limited sensitivity, reproducibility issues due to RNA isolation and reverse transcription steps, and the need for specific primer and probe design for each target, which complicates multiplex analysis and increases workload.
Innovation Solution
The method involves capturing surrogate nucleic acids associated with target nucleic acids, amplifying the surrogate nucleic acids, and detecting them to indicate the presence or quantity of the target nucleic acids, bypassing the need for RNA isolation and reverse transcription, and allowing for multiplex detection with pre-designed surrogate amplicons and probes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If qPCR is used for nucleic acid detection, then detection sensitivity is improved, but reproducibility deteriorates due to RNA isolation and reverse transcription steps
Solution Approach 1:
The invention extracts and eliminates the problematic pre-analytical steps (RNA isolation and reverse transcription) from the qPCR workflow by using a surrogate nucleic acid system that allows direct PCR amplification from crude samples, thereby removing the sources of variability while preserving detection sensitivity
Solution Approach 2:
The invention creates a copy (surrogate nucleic acid) that represents the target nucleic acid but can be amplified without the problematic conversion steps. The surrogate serves as a proxy that maintains the quantitative relationship with the target while enabling more reliable amplification
2Measurement precision
If specific primer and probe design is performed for each target, then detection accuracy is improved, but device complexity and workload increase
Solution Approach 1:
The invention creates a universal surrogate nucleic acid system that can be used across multiple targets and applications. The surrogate design allows a single amplification system to serve multiple detection purposes, eliminating the need for separate primer and probe designs for each target
Solution Approach 2:
The invention separates the detection system into two functional components: the target-specific capture element and the universal amplification/detection element. This segmentation allows the complex target-specific work to be done once in designing the capture element, while the amplification system remains simple and reusable
3Adaptability or versatility
If RNA isolation and reverse transcription steps are performed, then target nucleic acid conversion is achieved, but loss of time and increased procedural complexity occur
Solution Approach 1:
The invention performs the conversion function in advance by designing the surrogate nucleic acid to be directly amplifiable by PCR without requiring reverse transcription. The surrogate is pre-configured with the necessary properties to serve as a PCR template, eliminating the need for time-consuming conversion steps during sample processing
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 enhances sensitivity, precision, and simplicity in nucleic acid detection, reducing the complexity of primer and probe design and validation, and improving reproducibility, especially for low copy number targets, while minimizing stochastic effects and background noise.
Implementation Method 1
hybridizing the first surrogate capture probe to the first target nucleic acid and to the nucleic acid comprising the first surrogate nucleic acid
Implementation Method 2
amplifying the captured first surrogate nucleic acid to provide amplified first surrogate nucleic acid
Data Source
AI summary
Methods for detecting and optionally quantitating one or more target nucleic acids are provided, in which a surrogate nucleic acid is captured to each target nucleic acid, amplified, and detected. Compositions, kit, and systems related to the methods are also described.