Isothermal Nucleic Acid Amplification for Rapid Multiplexing

Solution Overview

Problem

Current nucleic acid amplification techniques, including PCR and isothermal methods, face challenges such as time-consuming results, requirement for specialized laboratories, and limited availability of fast and reliable multiplexing tests for diagnosing 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, allowing for simultaneous amplification of multiple target sequences in a portable device, enabling rapid and reliable diagnosis of infectious agents in various environments, including home settings, by utilizing recombinase, single-strand binding proteins, and thermostable DNA polymerases at optimized temperatures.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If PCR methods are used for nucleic acid amplification, then sensitivity and specificity are improved, but time-to-result and device portability deteriorate due to requirement for thermal cycling equipment and specialized laboratories

Engineering Contradiction:

Improvediagnostic accuracyVSAvoidtime-to-result

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The patent changes the temperature parameter from cyclic variation (PCR) to constant isothermal conditions (37-42°C), enabling the use of thermostable enzymes that function optimally at this constant temperature. This eliminates the need for complex thermal cycling equipment while maintaining amplification efficiency and diagnostic accuracy.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent replaces the mechanical thermal cycling system with a simple heating block or water bath capable of maintaining a constant temperature. This substitution dramatically simplifies the equipment requirements, enabling portability and point-of-care testing while preserving the sensitivity and specificity of nucleic acid amplification.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Reliability

If PCR methods are used for nucleic acid amplification, then sensitivity and specificity are improved, but device portability and ease of operation deteriorate due to requirement for specialized laboratories and thermal cycling equipment

Engineering Contradiction:

Improvediagnostic accuracyVSAvoidoperational simplicity

Core Design Contradiction:

ReliabilityVSEase of operation

Solution Approach 1:

The patent changes the temperature parameter from cyclic variation (PCR) to constant isothermal conditions (37-42°C), enabling the use of thermostable enzymes that function optimally at this constant temperature. This eliminates the need for complex thermal cycling equipment while maintaining amplification efficiency and diagnostic accuracy.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent replaces the mechanical thermal cycling system with a simple heating block or water bath capable of maintaining a constant temperature. This substitution dramatically simplifies the equipment requirements, enabling portability and point-of-care testing while preserving the sensitivity and specificity of nucleic acid amplification.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

3Ease of operation

If traditional isothermal methods are used, then operational simplicity is improved, but sensitivity and specificity deteriorate compared to PCR methods

Engineering Contradiction:

Improveoperational simplicityVSAvoiddiagnostic accuracy

Core Design Contradiction:

Ease of operationVSReliability

Solution Approach 1:

The patent changes the temperature parameter from cyclic variation (PCR) to constant isothermal conditions (37-42°C), enabling the use of thermostable enzymes that function optimally at this constant temperature. This eliminates the need for complex thermal cycling equipment while maintaining amplification efficiency and diagnostic accuracy.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent employs a composite enzymatic system comprising thermostable recombinase, thermostable DNA polymerase, and single-strand binding proteins that work synergistically at constant temperature. This composite approach combines the operational simplicity of isothermal methods with the high sensitivity and specificity traditionally associated with PCR.

Inventive Principle:

Principle #40Composite materials

4Adaptability or versatility

If multiplexing tests are implemented for diagnosing multiple infectious agents, then diagnostic comprehensiveness is improved, but test complexity and time-to-result worsen

Engineering Contradiction:

Improvemultiplexing capabilityVSAvoidtime-to-result

Core Design Contradiction:

Adaptability or versatilityVSLoss of time

Solution Approach 1:

The patent merges multiple target-specific primer sets into a single isothermal reaction mixture, allowing simultaneous amplification of multiple pathogens. The constant temperature condition enables all primer sets to function concurrently without interference, providing comprehensive multiplexing in a single test that delivers results in under 20 minutes.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent changes the temperature parameter from cyclic variation (PCR) to constant isothermal conditions (37-42°C), enabling the use of thermostable enzymes that function optimally at this constant temperature. This eliminates the need for complex thermal cycling equipment while maintaining amplification efficiency and diagnostic accuracy.

Inventive Principle:

Principle #35Parameter changes

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 provides fast and specific amplification of multiple nucleic acid sequences in under 20 minutes, reducing the risk of cross-contamination and enabling immediate action for infectious disease diagnosis, even in non-laboratory settings, with improved sensitivity and specificity compared to traditional methods.

Implementation Method 1

providing (i) at least one recombinase

Methodology Applied

Scientific EffectRecombinase-mediated strand invasion: Enzyme

Implementation Method 2

at least one DNA polymerase, preferably a strand displacement polymerase

Methodology Applied

Scientific EffectDNA polymerization: Enzyme

Implementation Method 3

at least one single-strand binding protein

Methodology Applied

Scientific EffectSingle-strand binding: Enzyme

Implementation Method 4

providing at least one reverse transcriptase

Methodology Applied

Scientific EffectReverse transcription: Enzyme

Data Source

PatentUS20230383338A1Isothermal nucleic acid amplification methods for point-of-need diagnosis

Publication Date: 2023.11.30 MIDGE MEDICAL GMBH

AI summary

The present invention relates to a fast and optionally multiplexing or multimeric method for 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.