Multiplex PCR Detection of Uveitis Pathogens From Small Eye Samples
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Solution Overview
Problem
The limited amount of anterior chamber fluid collected for uveitis diagnosis is insufficient for detecting multiple pathogens, and existing methods are prone to human error and inefficiency in pathogen detection.
Innovation Solution
A method involving mixing a sample with a PCR buffer containing a surfactant, adding it to a tube strip with DNA polymerase and PCR primer pairs, and detecting PCR products, which allows simultaneous detection of multiple pathogens without nucleic acid extraction, using a kit with a tube strip containing PCR reagents and fluorescent dyes for accurate results.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If nucleic acid extraction is performed from a small amount of anterior chamber fluid, then pathogen detection can be conducted, but sample loss occurs during the extraction operation step making it impossible to secure sufficient sample for detecting multiple pathogens
Solution Approach 1:
The invention extracts only the necessary component (nucleic acid) directly in the reaction tube without performing a separate extraction step. The PCR buffer with surfactant directly lyses cells and releases nucleic acid into the reaction mixture, eliminating sample loss during transfer and extraction operations while maintaining detection capability
Solution Approach 2:
The invention merges the nucleic acid extraction step with the PCR reaction setup step. By adding PCR buffer containing surfactant directly to the sample in the reaction tube, cell lysis and nucleic acid release occur in situ, combining what were previously separate operations into one integrated process that prevents sample loss
2Adaptability or versatility
If multiple PCR reagents and primers are prepared for detecting each pathogen individually, then comprehensive pathogen detection can be achieved, but human error such as incorrect reagent insertion increases and detection efficiency decreases
Solution Approach 1:
The invention creates a universal PCR buffer formulation containing surfactant that works for all pathogen detection targets simultaneously. This single buffer composition handles cell lysis and nucleic acid release for all pathogens, eliminating the need for separate reagent preparations for each pathogen and reducing operational complexity
Solution Approach 2:
The invention segments the detection process by using separate reaction tubes for different pathogen targets, each with its specific primer set, while using a common universal buffer formulation. This allows comprehensive pathogen detection through parallel processing of multiple targets without requiring complex integrated reagent systems
3Object-affected harmful factors
If a small amount of anterior chamber fluid is collected for genetic testing, then minimal patient discomfort is caused, but the sample amount becomes insufficient for detecting multiple pathogens
Solution Approach 1:
The invention changes the chemical parameters of the reaction buffer by incorporating surfactant, which dramatically improves cell lysis efficiency and nucleic acid release from small sample volumes. This parameter change allows sufficient nucleic acid extraction from minimal sample (100 μL or less) to support multiple pathogen detections without requiring larger sample collection
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
Enables rapid and accurate detection of multiple pathogens from a small sample volume, minimizing sample loss and human error, and ensuring efficient PCR performance.
Implementation Method 1
a step of mixing the sample with a PCR buffer containing a surfactant
Implementation Method 2
each tube contains DNA polymerase and one or more types of PCR primer pairs; a step of detecting the PCR product generated in the tube
Implementation Method 3
a kit with a tube strip containing PCR reagents and fluorescent dyes for accurate results
Data Source
AI summary
The present invention relates to a method for simultaneously detecting a plurality of pathogens from biologically-derived samples, and a kit for carrying out the method. Specifically, the present invention relates to a method for simultaneously detecting a plurality of pathogens that cause infectious uveitis, one of eye infections from samples such as anterior chamber fluid or vitreous by polymerase chain reaction (PCR), and a kit for carrying out the method.