Lysis Composition with Polyanionic Polymer for Inhibitor Neutralization
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Solution Overview
Problem
Current methods for analyzing nucleic acids in biological samples require costly and time-consuming purification steps, and existing lysis buffers often fail to effectively neutralize PCR inhibitors, leading to suboptimal performance in amplification reactions, especially when processing large sample volumes.
Innovation Solution
A method involving the use of a lysis composition containing at least one non-ionic surfactant and a water-soluble polyanionic polymer, such as polyacrylic acid, which efficiently neutralizes amplification inhibitors by complexing them during the lysis process, allowing the direct use of the lysate in nucleic acid amplification reactions without prior purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a conventional lysis buffer is used, then the sample can be lysed, but PCR inhibitors are not effectively neutralized, leading to suboptimal amplification performance
Solution Approach 1:
The patent introduces PVP (polyvinylpyrrolidone) as an intermediary substance that mediates between the PCR inhibitors present in the lysate and the amplification reaction. PVP acts as a trapping agent that selectively binds to inhibitors such as heme, bilirubin, and other PCR-inhibiting substances, thereby protecting the amplification reaction from their harmful effects while allowing the desired nucleic acid amplification to proceed with improved reliability
Solution Approach 2:
The patent modifies the chemical composition parameters of the lysis buffer by incorporating PVP at specific concentrations (0.01-5% w/v). This parameter change transforms the lysis buffer from a conventional formulation that merely lyses cells to an enhanced formulation that simultaneously neutralizes inhibitors, thereby improving amplification performance without requiring separate purification steps
2Reliability
If purification steps are performed to remove inhibitors, then amplification performance improves, but time and costs increase
Solution Approach 1:
The patent merges two previously separate processes into one unified step: cell lysis and inhibitor removal. By incorporating PVP into the lysis buffer, the buffer simultaneously performs cell lysis and inhibitor neutralization in a single operation, eliminating the need for separate purification steps and thereby reducing processing time while maintaining amplification performance
Solution Approach 2:
The lysis buffer is designed with multi-functionality, serving both to lyse cells and to remove inhibitors. The inclusion of PVP赋予 the buffer the additional function of inhibitor trapping, making it a universal solution that addresses both cell disruption and contamination removal in one reagent system, thus saving time and simplifying the workflow
3Measurement precision
If larger amounts of lysed sample are transferred to the amplification reaction, then sensitivity improves, but inhibitor interference increases
Solution Approach 1:
PVP serves as a mediator that allows larger sample volumes to be used in the amplification reaction. By binding to inhibitors in proportion to their concentration, PVP ensures that even when larger amounts of lysate are added to improve sensitivity, the inhibitors are simultaneously neutralized, maintaining optimal amplification conditions throughout
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 improves the performance of nucleic acid amplification reactions by effectively reducing inhibitor interference, enabling the use of larger sample volumes without significant inhibition, thus enhancing sensitivity and reducing the need for costly purification steps.
Implementation Method 1
a water-soluble polyanionic polymer, such as polyacrylic acid, which efficiently neutralizes amplification inhibitors by complexing them during the lysis process
Implementation Method 2
a lysis composition containing at least one non-ionic surfactant and a water-soluble polyanionic polymer
Data Source
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AI summary
The present invention pertains to a method for lysing a sample to provide a lysis mixture that is suitable for being directly used in a nucleic acid analysis method, wherein the sample is contacted with (a) at least one non-ionic surfactant, (b) at least one water-soluble polyanionic polymer, to provide a lysis mixture. The obtained lysis mixture can be used directly in an amplification reaction. The comprised water-soluble polyanionic polymer efficiently improves the amplification results by complexing PCR inhibitors that are released from the sample and are comprised in the lysis mixture.