Paraffin Separator for Single-Tube PCR Reagent Isolation
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Solution Overview
Problem
Existing nucleic acid amplification methods, such as PCR, require complex preparation processes, are prone to contamination, and demand high technical skills, making them unsuitable for high-throughput and rapid on-site testing, and existing sealing technologies have limitations in temperature sensitivity and dissolution time affecting reagent performance.
Innovation Solution
A paraffin separating agent is used to separate buffer solution, primer-probe mixture, and enzyme solution in a single container, which can be mixed by applying an external force like centrifugation, minimizing steps to sample addition and tube capping, while maintaining reagent performance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If buffer solution and enzymes are stored separately in different tubes, then the stability of primers and probe is maintained and enzymes have long-term shelf-life, but the preparation process becomes complex and requires multiple steps of mixing and aliquoting
Solution Approach 1:
The patent divides the reaction system into separate compartments within a single tube using a separating agent. The buffer solution, enzymes, primers, and probes are stored in separate sections that remain isolated during storage and transport, maintaining their individual stability. When needed, the separating agent is removed or degraded to allow mixing of all components in one tube, eliminating the need for multiple separate tubes and complex aliquoting procedures.
Solution Approach 2:
The patent nests multiple reagent components within a single tube structure, with the separating agent creating distinct zones or compartments. This allows multiple substances to be contained in one tube while remaining separated, effectively combining the benefits of separate storage with the convenience of a single-tube system.
2Ease of operation
If buffer solution and enzymes are mixed and aliquoted before use, then the preparation process is simplified, but the reaction system becomes prone to contamination and errors between batches
Solution Approach 1:
The patent uses a separating agent to create physical barriers between different reagent components within a single tube. This segmentation allows the tube to be opened once and all components to be mixed together, eliminating the need for multiple opening/closing cycles that increase contamination risk. The separating agent prevents cross-contamination between different reagent zones while maintaining a single-tube format that is easy to operate.
3Ease of operation
If sealing layers with different melting points are used to seal reaction reagents, then the reaction process can be controlled through temperature, but the dissolution of sealing layer takes time and affects reagent performance
Solution Approach 1:
The patent extracts the separating function from the sealing layer concept. Instead of using a sealing layer that must dissolve to release reagents, the patent uses a separating agent that can be physically removed or degraded without requiring prolonged heating. This eliminates the time-consuming dissolution step while maintaining the ability to control when reagents become accessible.
Solution Approach 2:
The patent changes the parameter of how separation is achieved - from temperature-based sealing layer dissolution to a separating agent that can be removed or degraded through alternative means (such as enzymatic degradation or physical removal). This parameter change eliminates the time loss associated with heating and dissolving sealing layers while maintaining reagent stability during storage.
4Reliability
If multiple steps of solution addition, mixing and tube cap opening are required, then reagent separation is achieved, but the workload increases and technical skill requirements increase
Solution Approach 1:
The patent merges multiple reagent components into a single tube system with internal separation provided by the separating agent. This allows all reagents to be accessed and mixed in one tube after a single cap opening, eliminating the need for multiple separate tube openings and transfers. The separating agent maintains reagent separation during storage while allowing easy mixing when needed.
Solution Approach 2:
The separating agent is designed to automatically separate reagents during storage and transport without requiring external intervention. When needed, the separating agent can be easily removed or degraded to allow mixing, making the system self-sufficient and eliminating the need for complex manual separation procedures.
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
Simplifies the nucleic acid amplification process, reduces contamination risk, and maintains reagent performance by using a paraffin separating agent that withstands transportation and mixing conditions without affecting PCR results.
Implementation Method 1
A paraffin separating agent is used to separate buffer solution, primer-probe mixture, and enzyme solution in a single container
Implementation Method 2
which can be mixed by applying an external force like centrifugation
Data Source
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AI summary
The present invention provides a method for separating a nucleic acid amplification system and a separating agent. The method utilizes a separating layer to separate the nucleic acid amplification system in the same container; and the separating layer can be ruptured by applying an external force, to realize mixing of the nucleic acid amplification system. The separating agent includes polyethylene wax, solid paraffin and liquid paraffin. The separating agent can effectively separate the buffer solution, primer-probe mixed solution and enzyme solution in the nucleic acid amplification system in a same container. When in use, only the sample to be tested is added, and after capping, an external force such as a centrifugal force is applied to rupture the separating agent, so that the nucleic acid amplification system can be mixed, and then directly subjected to on-computer test. By using the method and the separating agent of the present invention, only one step of sample addition and tube cap opening are needed, operations are minimized, the chance that the amplification reaction system contamination is reduced, and technical literacy requirements for operators are also reduced. Moreover, the separating agent and the method of operation of the present invention do not affect the PCR performance.