Multiplex PCR Quantification Using Internal Reference Standards
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Solution Overview
Problem
Current methods for detecting and quantifying microbial nucleic acids, such as HIV, Hepatitis-B, and HCV, using PCR face challenges like false-negative results due to variable sequence regions and low viral loads, and external calibration methods are prone to errors from extraction and inhibition issues.
Innovation Solution
A method involving the simultaneous amplification and detection of multiple sequence portions of microbial nucleic acids along with internal quantitative references in the same reaction mixture, reducing the risk of underquantitation and minimizing sensitivity variability, while using internal standards to account for sample-specific inhibitions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If external calibration is used for quantifying microbial nucleic acids, then quantification can be performed, but false-negative results occur due to extraction variability and inhibition issues not being accounted for
Solution Approach 1:
An internal quantitative reference nucleic acid is introduced as an intermediary element that co-amplifies with the target microbial nucleic acid throughout the entire process. This reference serves as a built-in control that accounts for extraction efficiency and inhibition effects, allowing accurate quantification without being affected by sample-specific variables. The reference nucleic acid acts as a mediator between the target and the detection system, providing a reliable basis for quantification.
Solution Approach 2:
The quantification process is segmented into two independent components: the target microbial nucleic acid and the internal quantitative reference nucleic acid. By separating the quantification function into distinct segments that can be independently tracked and compared, the method eliminates the need for external calibration while maintaining accuracy. Each segment is amplified and detected separately, allowing for precise relative quantification.
2Reliability
If multiple sequence portions are amplified by PCR, then detection reliability improves, but the complexity of the assay increases
Solution Approach 1:
Multiple sequence portions and the internal quantitative reference are merged into a single reaction mixture that undergoes simultaneous amplification. By combining multiple detection targets and the reference element in one PCR reaction, the method maintains high detection reliability through multi-target monitoring while minimizing assay complexity by eliminating the need for separate reactions for each target.
Solution Approach 2:
The PCR amplification system is designed with universal functionality to handle multiple sequence portions and the internal reference simultaneously. A single set of amplification conditions and a single reaction mixture serve multiple purposes: detecting different sequence portions and quantifying the target through the reference, thereby reducing overall assay complexity while maintaining reliability.
3Ease of manufacture
If a single sequence portion is targeted, then the assay is simple, but false-negative results increase due to mutations in variable sequence regions
Solution Approach 1:
The target microbial genome is segmented into multiple sequence portions, each targeted by specific primers. This segmentation allows the assay to monitor multiple regions simultaneously, reducing the impact of mutations in any single region. The internal quantitative reference provides an additional segment for comparison, maintaining assay simplicity while improving reliability through multi-point monitoring.
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 significantly reduces the risk of false-negative results and improves the accuracy of viral load quantification by leveraging multiple sequence portions and internal standards, enhancing sensitivity and reliability in detecting and quantifying microbial nucleic acids.
Implementation Method 1
The amplification is performed most commonly with the polymerase chain reaction which specifically amplifies target nucleic acids to detectable amounts
Implementation Method 2
Automated systems for PCR-based analysis often make use of real-time detection of product amplification during the PCR process. Key to such methods is the use of modified oligonucleotides carrying reporter groups or labels
Data Source
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AI summary
The present invention relates to new methods and uses for the detection and quantification of microbial nucleic acids employing an internal quantitative reference. Preferred methods are based on the amplification of nucleic acids, preferably the polymerase chain reaction. Further provided are kits comprising components for performing said methods and uses. Moreover, an analytical system for advantageously performing the method according to the invention is disclosed.