Alkaline Nucleic Acid Extraction Neutralization in PCR
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Conventional nucleic acid extraction methods, such as the Chelex-100 boiling method and alkaline lysis method, require manual neutralization steps, which are cumbersome and prone to errors, affecting PCR amplification efficiency and detection sensitivity.
Innovation Solution
A method that uses a strong alkaline nucleic acid extraction solution to extract nucleic acids, followed by direct addition to a PCR reaction solution with a lower pH for neutralization, eliminating the need for manual neutralization and allowing simultaneous amplification and detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If manual neutralization step is added after Chelex-100 boiling method or alkaline lysis method, then PCR amplification efficiency is improved, but operation complexity increases and detection time is extended
Solution Approach 1:
The patent combines the neutralization step with the PCR reaction setup by formulating the PCR reaction solution with buffering capacity to simultaneously perform neutralization and provide optimal pH for PCR amplification. This eliminates the separate manual neutralization step while maintaining PCR efficiency.
Solution Approach 2:
The PCR reaction solution is designed to automatically neutralize the alkaline extracted nucleic acid solution through its buffering components, eliminating the need for operator intervention. The system self-regulates the pH to suitable levels for PCR amplification.
2Reliability
If manual neutralization step is added after Chelex-100 boiling method or alkaline lysis method, then PCR amplification efficiency is improved, but detection time is extended
Solution Approach 1:
The patent merges the neutralization process with the PCR reaction initialization, so that the buffering action occurs concurrently with the setup of the PCR reaction conditions, eliminating sequential time loss.
Solution Approach 2:
The PCR reaction solution is pre-formulated with appropriate buffering capacity before use, so that when the alkaline extracted nucleic acid is added, neutralization begins immediately without requiring separate preparation or addition of neutralization reagents.
3Manufacturing precision
If operator manually adds neutralization solution, then pH control is improved, but human error risk increases
Solution Approach 1:
The system automatically controls pH through the buffering capacity of the pre-formulated PCR reaction solution, eliminating dependence on operator action. The pH regulation becomes a built-in property of the reaction mixture rather than a manual step.
Solution Approach 2:
The buffering system in the PCR reaction solution provides automatic feedback control of pH, resisting changes and maintaining optimal pH range for PCR amplification without requiring measurement or adjustment by the operator.
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 approach significantly reduces detection time, minimizes manual errors, and enhances the efficiency, sensitivity, and accuracy of PCR amplification and nucleic acid detection.
Implementation Method 1
using a strong alkaline nucleic acid extraction solution to extract the nucleic acid in a sample
Implementation Method 2
followed by direct addition to a PCR reaction solution with a lower pH for neutralization
Data Source
Figure 1
Figure 2
Figure 3
AI summary
Provided are a reagent, method, and test kit for measuring sample nucleic acid. A strong alkaline nucleic acid extraction solution (the pH value being 10.5 to 12.5) is used to extract the nucleic acid in the sample; add directly the extracted nucleic acid into a PCR reaction solution (the pH value being 8.0 to 9.0) for neutralization; at the same time, carry out nucleic acid amplification and measurement. The present invention can measure the nucleic acid in different types of samples such as blood, blood plasma, whole blood, genital tract secretion, sputum, or urine. In the present invention, the extracted sample nucleic acid is neutralized in a PCR reaction solution instead of being neutralized during nucleic acid extraction, thereby effectively enhancing the nucleic acid extraction efficiency and PCR amplification efficiency, and substantially improving the accuracy and sensitivity of the sample nucleic acid measurement.