Environmental Nucleic Acid Pretreatment with EDTA and Heat
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Solution Overview
Problem
Existing methods for nucleic acid detection in environmental samples, such as sewage water and rivers, are cumbersome, time-consuming, and costly, and are susceptible to the inhibitory effects of nucleases present in these samples, particularly for RNA viruses.
Innovation Solution
A pretreatment method involving the use of ethylenediaminetetraacetic acid (EDTA) in a composition, combined with heat treatment at 90°C or higher, to stabilize nucleic acids and inhibit nuclease activity, followed by rapid heating and subsequent nucleic acid detection using RT-PCR or PCR.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If commercial nucleic acid extraction kits are used to extract viral nucleic acids from samples, then extraction purity is improved, but pretreatment time increases to approximately 1 hour
Solution Approach 1:
The patent extracts and isolates the essential function of nucleic acid extraction from complex commercial kits, using only the core components (buffer solution with specific pH, metal ions, and reducing agents) to achieve purification without the time-consuming procedures of commercial kits. This selective extraction of functional elements resolves the contradiction between purity and time.
Solution Approach 2:
The patent optimizes specific parameters including buffer pH (6.0-9.0), metal ion concentrations (0.01-10 mM), and reducing agent concentrations (0.1-10 mM) to achieve effective nucleic acid extraction and stabilization within minutes. By precisely controlling these parameters, the method attains commercial-kit-level purity without the hour-long processing time.
2Measurement precision
If samples are collected from many measurement points in the environment for continuous measurement, then measurement coverage is improved, but pretreatment complexity and time increase
Solution Approach 1:
The patent creates a universal pretreatment method using a single buffer solution composition that effectively handles diverse environmental samples (sewage water, river water, soil extracts) without requiring sample-specific protocols. This multi-functional approach enables continuous measurement across many measurement points while maintaining simple, consistent pretreatment procedures.
Solution Approach 2:
The patent segments the complex pretreatment process into simple, sequential steps: (1) adding buffer solution containing metal ions and reducing agents, (2) brief incubation, and (3) direct PCR amplification. This segmentation eliminates complex procedures while maintaining effectiveness across various environmental samples.
3Measurement precision
If nucleic acids are extracted and purified using conventional methods, then detection accuracy is improved, but the process becomes time-consuming and costly
Solution Approach 1:
The patent merges the pretreatment and PCR amplification steps into a single reaction tube, eliminating the need for separate purification and concentration steps. The buffer solution simultaneously performs nucleic acid extraction, stabilization, and preparation for amplification, achieving detection accuracy comparable to conventional methods while reducing total time to under 1 hour including PCR.
Solution Approach 2:
The patent introduces a specially formulated buffer solution containing metal ions (Ca²⁺, Mg²⁺, Mn²⁺, Zn²⁺ at 0.01-10 mM) and reducing agents (DTT, β-mercaptoethanol, glutathione at 0.1-10 mM) as an intermediary that stabilizes nucleic acids and protects against degradation during the simplified extraction process, maintaining detection accuracy without complex purification.
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, accurate, and sensitive detection of nucleic acids in environmental samples, reducing the time required to under 4 hours and minimizing the impact of sample contaminants.
Implementation Method 1
a step of mixing a sample with a composition for pretreatment comprising ethylenediaminetetraacetic acid (EDTA)
Implementation Method 2
a step of heating the sample mixed in step i) using a heating means maintained at 90°C or higher
Data Source
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AI summary
The present disclosure concerns detection of a target nucleic acid comprised in a sample, in particular, a water sample in the environment, such as treated sewage water or rivers, in a more convenient manner with higher accuracy. A target sample of nucleic acid detection is mixed with a composition for pretreatment comprising EDTA and the resultant is then heated using a heating means maintained at 90°C or higher.