Multiplex Nucleic Acid Detection Using Embedded Solid Support Probes
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Solution Overview
Problem
Current multiplex assays for detecting respiratory viruses, such as those causing avian flu, SARS, and COVID-19, face challenges including low sensitivity, high background noise, and the need for multiple washing steps, which increase time and cost, and often require skilled personnel and specific conditions for RNA controls, limiting their accessibility and accuracy.
Innovation Solution
A composition and method using a plasmid with target nucleic acid sequences, a passive reference dye, and specific DNA primers and probes for PCR, which reduces background noise and increases sensitivity, stability, and accessibility by using a stable plasmid control and total human RNA as a non-viral extraction control, allowing for simultaneous detection of multiple viruses like influenza A, B, and SARS-CoV-2.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional multiplex PCR assays are used to detect multiple respiratory viruses, then the ability to detect multiple viruses simultaneously is improved, but the background noise increases and sensitivity decreases
Solution Approach 1:
The patent extracts and removes the problematic washing steps from the traditional multiplex PCR workflow. By using a solid support with embedded probes that bind directly to amplified viral sequences without requiring washing, the method eliminates the source of background noise and signal loss while maintaining the ability to detect multiple viruses simultaneously.
Solution Approach 2:
The patent introduces a solid support as an intermediary carrier that contains embedded complementary DNA probes. This solid support mediates the detection process by capturing amplified viral sequences directly during the PCR amplification phase, eliminating the need for separate hybridization and washing steps, thereby reducing background noise and improving sensitivity.
2Manufacturing precision
If multiple washing steps are added to traditional PCR detection methods, then the specificity of virus detection is improved, but the time required and handling complexity increase
Solution Approach 1:
The patent extracts and eliminates the multiple washing steps from the traditional detection workflow. The solid support with embedded probes allows specific viral sequence detection to occur directly during PCR amplification without requiring subsequent washing steps, thereby maintaining specificity while dramatically reducing assay time and handling complexity.
Solution Approach 2:
The patent performs the specific binding action in advance by embedding complementary DNA probes into the solid support before the PCR reaction begins. These pre-positioned probes are ready to capture amplified viral sequences immediately during amplification, eliminating the need for post-amplification washing steps to achieve specificity.
3Measurement precision
If RNA controls are used in PCR assays, then the accuracy of viral detection is improved, but the stability and ease of use decrease due to manipulation requirements
Solution Approach 1:
The patent creates a stable copy of the RNA control function using DNA. Instead of using fragile RNA controls that require special handling, the invention uses stable DNA probes embedded in the solid support that can be stored long-term without degradation. These DNA copies perform the same control function by binding to complementary amplified sequences, maintaining detection accuracy while eliminating stability and handling issues.
4Productivity
If traditional immunoassays are used for virus detection, then the cost is reduced and speed is improved, but the ability to detect multiple virus species is limited
Solution Approach 1:
The patent creates a universal detection platform where a single solid support can detect multiple different virus species simultaneously. By embedding multiple different complementary DNA probes on the same solid support, the system achieves multi-functionality, allowing detection of various respiratory viruses in a single PCR reaction without the cost and complexity of separate immunoassays for each virus.
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
The solution provides a more accurate, sensitive, and cost-effective method for detecting multiple respiratory viruses with reduced variability and labor costs, enhancing the ability to diagnose respiratory infections efficiently and effectively, especially in non-specialized settings.
Implementation Method 1
a composition for detecting more than one target nucleic acid sequence in a nucleic acid sample by polymerase chain reaction (PCR)
Data Source
AI summary
A composition for detecting more than one target nucleic acid sequence in a nucleic acid sample by polymerase chain reaction (PCR) is provided. Also provided is a kit comprising the above composition with additional reagents for performing PCR using the plasmid, dye, primers and probes to detect the more than one target nucleic acid sequence. Additionally provided is a method for detecting more than one target nucleic acid sequence in a nucleic acid sample by polymerase chain reaction (PCR).