Complexing Agent for Nucleic Acid Detection in Transport Medium
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The use of Amies medium in transporting biological samples can lead to undesirable binding of free nucleic acids to sparingly soluble alkaline earth metal salts, making them unavailable for PCR detection due to their emulsifying properties, and establishing chaotropic conditions for nucleic acid binding on filters is challenging, resulting in poor detection sensitivity.
Innovation Solution
Adding a complexing agent that forms soluble complexes with calcium and magnesium ions, adjusting the pH to 5-6 with buffers, and using a dual-filter system with a porous volume filter and a membrane filter to isolate and lyse cells, allowing for effective nucleic acid release and detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Amies medium is used for transporting biological samples, then bacterial samples can be transported without damage, but nucleic acids bind to alkaline earth metal salts making them unavailable for PCR detection
Solution Approach 1:
The harmful effect of alkaline earth metal salts binding nucleic acids is eliminated by extracting/removing these salts from the system through complexation with EDTA. The EDTA forms stable complexes with calcium and magnesium ions, effectively removing them from the solution and preventing their interaction with nucleic acids, thus resolving the contradiction between maintaining sample integrity and preserving nucleic acid availability.
Solution Approach 2:
EDTA acts as an intermediary substance that mediates between the Amies medium and nucleic acids. It selectively binds to alkaline earth metal ions, preventing these ions from interacting with nucleic acids. This intermediary mechanism allows the Amies medium to maintain its protective function while EDTA simultaneously prevents nucleic acid binding, resolving the contradiction.
2Measurement precision
If chaotropic conditions are established for nucleic acid binding on filters, then detection sensitivity should improve, but it is challenging to achieve optimal conditions without losing cell integrity
Solution Approach 1:
The method performs preliminary actions in a specific sequence: first adding EDTA to remove alkaline earth metal salts and prevent nucleic acid binding, then proceeding with cell lysis and nucleic acid extraction. This preliminary removal of interfering substances creates optimal conditions for subsequent detection steps while maintaining cell integrity throughout the process, resolving the contradiction between detection sensitivity and cell integrity.
Solution Approach 2:
The method changes key parameters of the system: it adjusts the chemical environment by adding EDTA to complex metal ions, controls pH levels, and manages ionic strength. These parameter changes create optimal conditions for nucleic acid detection while preserving cell integrity, effectively resolving the contradiction between measurement precision and reliability.
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
Converts sparingly soluble alkaline earth metal salts into a dissolved form, preventing nucleic acid binding and enhancing detection sensitivity by maintaining cell integrity and facilitating PCR reactions.
Implementation Method 1
at least one complexing agent that forms complexes with alkaline earth metal ions, in particular with calcium ions and with magnesium ions, is added to the transport medium
Implementation Method 2
Use of a device for isolating cells that has, in succession, a porous volume filter and a membrane filter
Data Source
AI summary
The present disclosure relates to a method for treating a biological sample contained in a transport medium. At least one complexing agent is added to the transport medium, which complexing agent forms complexes with alkaline-earth metal ions. In the method, a device can be used which comprises, in succession, a porous volume filter and a membrane filter having a pore size in the range of 0.2 μm to 2.0 μm.