Simultaneous Microbial Nucleic Acid Detection and Quantification
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Solution Overview
Problem
Current molecular diagnostics for microbial nucleic acids, such as PCR-based methods, face challenges in simultaneously achieving accurate qualitative and quantitative detection due to the need for separate assays and internal controls, which can lead to false results and increased costs and complexity.
Innovation Solution
A method that uses a reaction mixture with a first control nucleic acid and/or a second control nucleic acid in different concentrations, along with specific primer pairs and probes, to simultaneously detect and quantify microbial nucleic acids, allowing for the same reagents to be used for both qualitative and quantitative assays, reducing the need for separate tests and improving assay validity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If separate qualitative and quantitative assays are used for microbial nucleic acid detection, then each assay can be optimized for its specific purpose, but the overall test complexity and cost increase
Solution Approach 1:
The patent combines separate qualitative and quantitative assays into a single integrated assay that simultaneously detects and quantifies microbial nucleic acids. The reaction mixture includes both a qualitative internal control nucleic acid and a quantitative standard nucleic acid, allowing both types of measurements to be performed in one test rather than requiring separate assays.
Solution Approach 2:
The assay design makes the reaction mixture universal by incorporating components that serve multiple functions: the qualitative internal control validates the reaction, the quantitative standard enables absolute quantification, and both are detected using the same PCR reagents and instrumentation. This multi-functional approach eliminates the need for separate specialized assays.
2Reliability
If a qualitative internal control nucleic acid is used at low concentration to ensure sensitivity, then false negatives are reduced, but the ability to perform quantitative measurement is compromised
Solution Approach 1:
The patent segments the control nucleic acids into two distinct components with different concentrations and functions: a qualitative internal control nucleic acid at low concentration (to ensure sensitivity and detect inhibition) and a quantitative standard nucleic acid at known higher concentration (to enable accurate quantification). Each segment is optimized for its specific purpose while both are present in the same reaction.
Solution Approach 2:
Different regions or components of the control system have different properties: the qualitative internal control is present at low concentration with specific sequence characteristics optimized for sensitivity, while the quantitative standard is present at higher concentration with characteristics optimized for accurate measurement. Each component's local quality is tailored to its specific function.
3Ease of manufacture
If external calibration is used for quantitative measurement, then standard curves can be created separately, but sample-specific effects such as extraction efficiency and inhibition are not accounted for
Solution Approach 1:
The quantitative standard nucleic acid acts as an intermediary that is added to each individual sample reaction mixture. This intermediary experiences the same sample-specific conditions (extraction efficiency, inhibition, matrix effects) as the target nucleic acid, allowing it to serve as an internal reference that accounts for these variations and enables accurate quantification despite sample-specific effects.
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 enables reliable simultaneous detection and quantification of microbial nucleic acids, reducing the burden on patients and healthcare systems by eliminating the need for multiple tests, while providing accurate results through the use of a common assay design and internal controls that function in both target-positive and target-negative reactions.
Implementation Method 1
The method is based on amplification of nucleic acids, for example the polymerase chain reaction
Implementation Method 2
two or more probes that hybridize to each of the sequences amplified by the one or more primer pairs, wherein the two or more probes are each labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety
Data Source
AI summary
The present invention relates to new methods and uses for the qualitative and quantitative detection of microbial nucleic acids using at least a first control nucleic acid, or a first and a second control nucleic acid in different concentrations. The method is based on amplification of nucleic acids, for example the polymerase chain reaction. Further provided are kits comprising components for performing said methods and uses.