Multiplex Isothermal Nucleic Acid Amplification for Rapid Diagnosis
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Solution Overview
Problem
Current nucleic acid amplification techniques, including PCR and isothermal methods, face challenges such as time-consuming processes, requirement for specialized laboratories, and limited availability of portable and reliable diagnostic tools for rapid detection of multiple infectious agents, particularly in non-laboratory settings, leading to delayed isolation and treatment of infected individuals.
Innovation Solution
A multiplex isothermal nucleic acid amplification method using recombinase polymerase amplification (RPA) with thermostable enzymes, enabling simultaneous amplification of multiple target sequences in a portable device, suitable for both laboratory and non-laboratory environments, which reduces the need for thermal cycling and enhances specificity and sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If PCR is used for nucleic acid amplification, then sensitivity and specificity are improved, but time-to-result and device portability deteriorate
Solution Approach 1:
The patent changes the temperature parameter from cyclic (PCR) to isothermal (constant 37-42°C), enabling rapid amplification without thermal cycling. This parameter change reduces time-to-result while maintaining diagnostic accuracy through optimized enzymatic reactions at constant temperature
Solution Approach 2:
The patent replaces the mechanical thermal cycling system with a biochemical isothermal amplification system using recombinase polymerase and auxiliary proteins. This substitution eliminates the need for complex thermal cycler equipment, enabling portable device implementation while maintaining sensitivity and specificity
2Reliability
If PCR is used for nucleic acid amplification, then sensitivity and specificity are improved, but device complexity and portability deteriorate
Solution Approach 1:
The patent replaces the mechanical thermal cycling system with a biochemical isothermal amplification system using recombinase polymerase and auxiliary proteins. This substitution eliminates the need for complex thermal cycler equipment, enabling portable device implementation while maintaining sensitivity and specificity
Solution Approach 2:
The patent employs a self-assembling recombinase polymerase complex that automatically binds to target DNA and initiates amplification without external control. The auxiliary proteins (SSB, helicase) autonomously perform their functions, eliminating the need for complex instrument control systems
3Adaptability or versatility
If multiplexing is implemented to detect multiple targets, then diagnostic comprehensiveness is improved, but test complexity and cost deteriorate
Solution Approach 1:
The patent uses a universal isothermal amplification system with recombinase polymerase that can detect multiple different DNA targets simultaneously in the same reaction well. Different fluorescent probes target different sequences, allowing one test to screen for multiple pathogens or genetic conditions without requiring separate tests for each target
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 method allows for rapid, reliable, and specific detection of multiple infectious agents in a single test, providing results within 20 minutes, reducing the risk of contamination and enabling immediate action, while also being cost-effective and user-friendly for non-experts.
Implementation Method 1
A mix is provided in a second chamber, which comprises a set of enzymes for performing an isothermal method of nucleic acid amplification
Data Source
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AI summary
The present invention relates to a multiplexing or multimeric method for fast isothermal amplification of nucleic acids, including DNA and RNA. Particularly, the invention relates to diagnostic methods for rapidly diagnosing, for example, at least two infectious agents, or at least two different targets in the same infectious agent, in a biological probe of interest. The invention further relates to a handheld and portable diagnostic system for performing the amplification method in a laboratory as well as in a non-laboratory environment. Further provided are suitable enzyme sequences, kits and uses of the method and the system.