Isothermal Nucleic Acid Amplification with Lyophilized Reagents

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Solution Overview

Problem

Conventional nucleic acid testing methods require specialized lab facilities and instruments, are prone to false positives due to DNA contamination, and have limitations in specificity and robustness, especially in field or resource-limited settings, and existing lyophilized formulations for nucleic acid amplification reagents are not stable at ambient temperatures and require complex rehydration processes.

Innovation Solution

A method for nucleic acid amplification using a self-sustained reaction at a temperature between 42°C and 50°C, which is isothermal, reducing the need for thermal cycling and allowing for rapid and specific amplification without specialized equipment, combined with lyophilized formulations that maintain enzyme stability and facilitate rapid rehydration at ambient temperatures.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If conventional RT-PCR methods are used for nucleic acid amplification, then high sequence-specific detection sensitivity is achieved, but specialized thermal cycling instruments and lab facilities are required

Engineering Contradiction:

Improvedetection sensitivityVSAvoidinstrument requirement

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent replaces the mechanical thermal cycling system with a biochemical isothermal amplification system. The self-sustained sequence replication reaction maintains constant temperature (40-50°C) and achieves amplification through enzymatic mechanisms rather than thermal denaturation cycles, eliminating the need for complex thermal cyclers while maintaining detection sensitivity

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The amplification system is self-sustaining through the use of self-priming oligonucleotides that contain promoter sequences. The RNA polymerase extends these primers autonomously without requiring external thermal cycling control, making the system self-regulating and suitable for field deployment without specialized instruments

Inventive Principle:

Principle #25Self-service

2Ease of operation

If transcription-based amplification methods are used, then isothermal conditions simplify the process, but specificity decreases and DNA contamination causes false positives

Engineering Contradiction:

Improveisothermal operationVSAvoidspecificity

Core Design Contradiction:

Ease of operationVSReliability

Solution Approach 1:

The oligonucleotide primers are segmented into functional domains: a promoter sequence region that binds RNA polymerase and an annealing region that specifically hybridizes to the target RNA. This segmentation allows the primer to distinguish between RNA targets and DNA contaminants, maintaining specificity under isothermal conditions

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The primer design implements local quality by creating distinct functional regions within the oligonucleotide. The promoter region provides universal binding for the polymerase enzyme while the sequence-specific annealing region provides target discrimination, enabling both ease of operation and high specificity simultaneously

Inventive Principle:

Principle #3Local quality

3Stability of the object's composition

If conventional lyophilized formulations are used for reagent storage, then stability is improved, but complex rehydration processes are required and ambient temperature stability is not achieved

Engineering Contradiction:

Improvereagent stabilityVSAvoidrehydration complexity

Core Design Contradiction:

Stability of the object's compositionVSEase of manufacture

Solution Approach 1:

The lyophilized formulation uses a composite protective matrix consisting of sugars (trehalose, sucrose, or lactose) combined with amino acids (glycine, alanine, or valine). This composite material provides superior protection to enzymes during lyophilization and storage, enabling stable storage at ambient temperatures and simple rehydration by adding only water without complex buffers or additives

Inventive Principle:

Principle #40Composite materials

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 method achieves faster and more specific nucleic acid amplification, is tolerant to temperature fluctuations, and allows for nucleic acid testing in field settings without the need for specialized facilities, with lyophilized formulations maintaining stability and activity for extended periods at ambient temperatures.

Implementation Method 1

amplification of a target nucleic acid by a self-sustained sequence replication reaction which is carried out under isothermal conditions at a temperature between 40° C. and 50° C.

Methodology Applied

Scientific EffectIsothermal amplification:

Implementation Method 2

amplification of a target nucleic acid by a self-sustained sequence replication reaction

Methodology Applied

Scientific EffectSelf-sustained sequence replication:

Implementation Method 3

lyophilised formulations for nucleic acid amplification reagents are not stable at ambient temperatures and require complex rehydration processes

Methodology Applied

Scientific EffectLyophilization: Freeze Drying

Data Source

PatentUS11447821B2Nucleic acid amplification and testing

Publication Date: 2022.09.20 CAMBRIDGE ENTERPRISE LTD

AI summary

Methods for amplifying a target nucleic acid by self-sustained amplification methods are described. The methods are designed, in particular, to be carried out without use of specialised lab facilities or instruments. Compositions, lyophilised formulations, and kits for carrying out the methods are also described.