Self-contained Nucleic Acid Analysis Pouch for Multiplex PCR
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Solution Overview
Problem
Traditional microbiology techniques for diagnosing infectious diseases are time-consuming, and while PCR has become a rapid diagnostic tool, it faces challenges with multiplex reactions and sample volume requirements, often necessitating extensive handling that can lead to contamination and inefficiencies.
Innovation Solution
The development of self-contained nucleic acid analysis pouches that integrate multiple processing zones for PCR, allowing for sample preparation, amplification, and detection within a single closed system, utilizing pneumatic pressure and pinch valves to manage reagents and samples efficiently, reducing handling and contamination risks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiplex PCR assays are run to diagnose multiple pathogens simultaneously, then diagnostic efficiency is improved, but reaction robustness deteriorates and product analysis becomes difficult
Solution Approach 1:
The patent divides the multiplex PCR process into two separate stages: a first PCR reaction and a second PCR reaction. The first reaction amplifies target sequences from multiple pathogens simultaneously, while the second reaction further amplifies specific products from the first reaction using nested primers. This segmentation maintains diagnostic efficiency by testing for multiple pathogens while improving reaction robustness through sequential, rather than simultaneous, amplification processes.
Solution Approach 2:
The patent implements nested PCR where the second PCR reaction uses primers that bind within the amplification products of the first PCR reaction. This nested approach allows specific pathogen products to be further amplified from the complex mixture of first reaction products, improving the reliability of detection while maintaining the ability to diagnose multiple pathogens. The nested design reduces non-specific amplification and improves product analysis clarity.
2Reliability
If secondary PCR reactions are nested within primary products to increase robustness, then reaction reliability is improved, but handling complexity and contamination risk increase
Solution Approach 1:
The patent combines the first and second PCR reactions into a single sealed microcentrifuge tube. Both reaction mixtures are added to the same tube, and thermal cycling is performed in sequence without opening the tube between reactions. This merging approach maintains reaction robustness through nested amplification while significantly reducing handling complexity and contamination risk by eliminating the need to transfer products between tubes.
Solution Approach 2:
The system performs both PCR reactions within the same sealed container using automated thermal cycling. The first reaction products serve directly as templates for the second reaction without manual intervention for product transfer. This self-service approach maintains high reaction robustness while minimizing human handling and potential contamination.
3Reliability
If nested PCR is performed with separate tube transfers, then amplification robustness is improved, but contamination risk and operational time increase
Solution Approach 1:
The patent combines both PCR reactions in a single sealed microcentrifuge tube, eliminating the need to transfer amplification products between tubes. The first reaction mixture is prepared in the tube, then the second reaction mixture is added to the same tube before a single thermal cycling program executes both reactions sequentially. This approach maintains amplification robustness through nested primer design while reducing operational time by eliminating transfer steps and associated contamination risks.
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 system enables robust and efficient multiplex PCR analysis with reduced handling, improving diagnostic speed and accuracy by integrating all necessary steps within a compact, sealed environment, thereby addressing the limitations of traditional methods.
Implementation Method 1
The instrument is configured to apply pneumatic pressure to move material within and between the zones
Implementation Method 2
pinch valves to manage reagents and samples efficiently
Data Source
Figure 1
Figure 2~2b
Figure 3~4
AI summary
The invention provides a system for delivering one or more liquids into a preselected array of a plurality wells comprising: a plurality of tubes, each tube having a first end in fluid communication with a reservoir, and a second end terminating in an orifice; a plurality of the reservoirs, the reservoirs provided in a predetermined configuration relative to one another; a print head operable to movably hold each orifice in a predetermined position such that the position of each orifice corresponds to a well in the preselected array of wells; a plurality of straws, each straw having a hollow opening connecting a first end to a second end, the first end fluidly connected to the first end of a corresponding tube and the second end removably in contact with a bottom portion of a corresponding reservoir; a metering device operable to urge a preselected amount of fluid from the second end of each straw through its corresponding tube, and out its corresponding orifice; an imaging system; and a computing device, wherein the computing device interfaces with the imaging system to accept or reject an array of drops on the print head, either prior to or subsequent to transfer to the array of wells.