Hydrogen Peroxide Nucleic Acid Extraction for Viscous Sample Liquefaction
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Solution Overview
Problem
Conventional methods for nucleic acid extraction from viscous samples like sputum, saliva, and pus samples result in significant nucleic acid loss due to strong alkaline environments and complex, time-consuming processes, leading to inefficient extraction and potential degradation of RNA.
Innovation Solution
A reagent kit utilizing a hydrogen peroxide solution with a molarity of 0.1-2 mol/L to liquefy samples, combined with a lysis solution containing surfactants and protein denaturation agents, reduces nucleic acid loss and improves extraction efficiency by releasing nucleic acids under mild conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If conventional sputum liquefaction methods (sodium hydroxide, DTT, or protease) are used, then the sputum can be liquefied, but nucleic acid loss occurs and the process is time-consuming or costly
Solution Approach 1:
The patent changes the chemical parameters of the liquefaction reagent by using hydrogen peroxide solution with specific concentration ranges (0.1-2 mol/L) instead of conventional strong alkalis like sodium hydroxide. This parameter change maintains effective liquefaction while creating a milder chemical environment that prevents nucleic acid degradation and loss.
Solution Approach 2:
The patent employs hydrogen peroxide, a cost-effective reagent that decomposes into water and oxygen, replacing expensive enzymes like protease or DTT. The reagent performs its liquefaction function and then decomposes harmlessly, eliminating the need for costly proteinase K treatment while achieving complete mucin degradation.
2Ease of operation
If strong alkaline environment is used for sputum liquefaction, then liquefaction is achieved, but nucleic acid degradation occurs
Solution Approach 1:
The patent fundamentally changes the pH parameter of the liquefaction environment by replacing strong alkaline reagents (pH 12-14) with hydrogen peroxide solution (pH near neutral). This parameter change enables effective liquefaction through oxidative degradation of mucin while maintaining nucleic acid integrity through the milder chemical environment.
Solution Approach 2:
The patent substitutes the chemical mechanism of strong base hydrolysis with the oxidative mechanism of hydrogen peroxide. Instead of using mechanical disruption or strong chemical hydrolysis that damages nucleic acids, the system uses oxidative degradation of disulfide bonds in mucin to achieve liquefaction while preserving biomolecule integrity.
3Ease of operation
If DTT or protease is used for sputum liquefaction, then mucin degradation is achieved, but cost increases and RNA loss occurs
Solution Approach 1:
The patent replaces expensive reagents (DTT at hundreds of yuan per bottle, proteinase K at thousands of yuan per bottle) with inexpensive hydrogen peroxide (a few yuan per bottle). The hydrogen peroxide performs mucin degradation through oxidation and then decomposes into harmless water and oxygen, providing a cost-effective alternative that eliminates the need for subsequent proteinase K treatment.
Solution Approach 2:
The patent utilizes the endogenous catalase present in sputum samples to catalyze the decomposition of hydrogen peroxide, generating oxygen bubbles that physically disrupt mucin structure. This self-service mechanism eliminates the need for external protease addition, reducing reagent costs while achieving complete liquefaction.
4Reliability
If conventional three-step processing (liquefaction, cell lysis, nucleic acid extraction) is used, then nucleic acid extraction is achieved, but processing time increases
Solution Approach 1:
The patent merges the conventional three-step processing (liquefaction, cell lysis, nucleic acid extraction) into a simplified two-step process. The hydrogen peroxide solution simultaneously performs both liquefaction of mucin and lysis of microbial cells, eliminating the need for separate proteinase K treatment and reducing total processing time while maintaining nucleic acid extraction efficiency.
Solution Approach 2:
The patent赋予 hydrogen peroxide solution multiple functions: it acts as a liquefaction reagent by degrading mucin, as a cell lysis reagent by oxidizing cell membranes, and as a disinfectant by killing pathogens. This multi-functionality consolidates multiple processing steps into one, significantly reducing processing time while ensuring complete nucleic acid extraction.
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 effectively liquefies viscous samples in a shorter duration, reduces nucleic acid loss, and enhances extraction yield and purity by using a hydrogen peroxide solution, surfactants, and optimized lysis procedures.
Implementation Method 1
After the hydrogen peroxide solution is added, substances in the microbial cells react with hydrogen peroxide to generate gas, and generated bubbles can destroy mucus or block-shaped samples, causing the samples to be completely liquefied
Implementation Method 2
For samples containing catalase and having high viscosity and easiness in agglomeration, such as sputum samples, saliva samples and pus samples, the sample can be fully liquefied by processing with the hydrogen peroxide in a suitable concentration for 0.5-3 min
Data Source
Figure 1~2
AI summary
Disclosed is a reagent kit and a nucleic acid extraction method. The kit is used for nucleic acid sample extraction. The kit comprises a hydrogen peroxide solution, and the molarity of the hydrogen peroxide solution is 0.1-2 mol/L. For certain nucleic acid samples, microbial cells included therein are wrapped by substances such as mucus or mucin and are difficult to release. After the hydrogen peroxide solution is added, substances in the microbial cells react with hydrogen peroxide to generate gas, and generated bubbles can destroy mucus or block-shaped samples, causing the samples to be completely liquefied, so as to release the microbial cells wrapped by the substances such as mucus or mucin, so that subsequent nucleic acid extraction steps can be smoothly carried out.