Colorimetric LAMP System with Lyophilized Reagents and Direct Lysis
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Solution Overview
Problem
Existing nucleic acid detection systems, particularly loop-mediated isothermal amplification (LAMP) technologies, face challenges such as high cost, complexity, reagent stability issues, and the need for sample purification, which limit their use in home-based settings and prolong diagnostic times.
Innovation Solution
A colorimetric LAMP system with an all-in-one lyophilized reaction mixture and an extraction-free lysis buffer, using a primer set, strand-displacing polymerase, and pH indicating dye, along with a direct lysis buffer containing chaotropic salts and detergents, allowing for rapid, sensitive, and specific diagnosis without the need for nucleic acid extraction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If colorimetric detection reagent is used with high concentration anti-freeze component (betaine and glycerol), then color change detection sensitivity is improved, but reagent stability deteriorates due to inability to freeze-dry
Solution Approach 1:
The patent changes the concentration parameters of anti-freeze components from high concentration (conventional) to optimized lower concentrations (0.1-1.0 M betaine, 0.05-0.5 M glycerol), enabling freeze-drying while maintaining sufficient color change detection sensitivity
Solution Approach 2:
The patent creates a composite reagent system combining colorimetric detection components with freeze-drying compatible formulation, integrating multiple functions (color change detection, enzyme stability, and freeze-drying capability) into a single stable composition
2Reliability
If traditional nucleic acid extraction and purification is performed, then sample purity is improved, but diagnostic time increases and sensitivity may decrease due to nucleic acid nicking
Solution Approach 1:
The patent removes the separate nucleic acid extraction and purification steps from the diagnostic workflow, using a direct lysis buffer that allows PCR amplification immediately from crude lysate, eliminating time-consuming purification procedures
Solution Approach 2:
The lysis buffer formulation is designed to self-cleanse the sample by suppressing inhibitor effects and protecting nucleic acids during lysis, eliminating the need for external purification interventions
3Stability of the object's composition
If separate lyophilization of PCR components (enzyme and primer) is performed, then reagent stability is improved, but manufacturing process complexity increases and duration is prolonged
Solution Approach 1:
The patent combines all PCR components (enzyme, primers, dNTPs, buffer, and anti-freeze agents) into a single lyophilized reagent formulation, enabling one-step manufacturing and simplifying the supply chain while maintaining stability
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
Enables rapid, sensitive, and specific diagnosis of viral genomic materials via naked eye detection, with reduced turnaround time, stability at room temperature, and compatibility for home-based use, maintaining sensitivity and specificity comparable to traditional methods.
Implementation Method 1
a pH indicating dye in a concentration ranged 0.08 to 0.3 mM
Implementation Method 2
The lyophilized colorimetric LAMP reaction mixture is rehydrated with the extraction-free lysis buffer to be ready for nucleic acid amplification and detection
Data Source
AI summary
A colorimetric LAMP system includes a colorimetric LAMP reaction mixture and an extraction-free lysis buffer. The colorimetric LAMP reaction mixture is all-in-one lyophilized and includes a primer set, a strand-displacing polymerase and deoxyribonucleoside triphosphates for amplifying a target sequence; a pH indicating dye in a concentration ranged 0.08 to 0.3 mM; and a lyoprotectant sugar in a concentration ranged 1 to 10% (w/v), wherein the lyoprotectant sugar is selected from the group consisting of trehalose, raffinose, dextran, mannitol and mixtures thereof. The extraction-free lysis buffer includes potassium chloride in a concentration ranged 10 to 50 mM; ammonium sulfate in a concentration ranged 10 to 50 mM; and a detergent in a concentration ranged 0.5 to 6% (w/v). The lyophilized colorimetric LAMP reaction mixture is rehydrated with the extraction-free lysis buffer to be ready for nucleic acid amplification and detection.