Nested PCR Panel for Rapid Pathogen Identification
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Solution Overview
Problem
Traditional microbiology techniques for diagnosing infectious diseases are time-consuming and often delayed by contamination issues in multiplex PCR and immuno-PCR assays, especially when dealing with low concentrations of pathogen nucleic acid and limited sample volumes.
Innovation Solution
A self-contained nucleic acid analysis system using disposable plastic pouches for nested PCR, which minimizes contamination and ensures robust amplification by amplifying conserved genes in a single reaction mixture and dividing it into secondary reactions with specific primers for each organism, allowing for simultaneous detection of multiple biological substances.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional microbiology techniques are used for pathogen diagnosis, then comprehensive pathogen identification can be achieved, but the diagnosis time is extended to days or weeks
Solution Approach 1:
The diagnostic process is segmented into two stages: a first-stage multiplex PCR that simultaneously amplifies multiple conserved gene targets, followed by a second-stage confirmation PCR. This segmentation allows rapid initial screening while maintaining accurate pathogen identification through sequential analysis.
Solution Approach 2:
The first-stage multiplex PCR performs preliminary amplification of multiple gene targets simultaneously, preparing amplicons that can be directly used in the second-stage confirmation PCR. This preliminary action eliminates the need for separate DNA extraction and amplification steps for each potential pathogen.
2Measurement precision
If large panels of PCR assays are run for each possible causative organism, then comprehensive pathogen detection is achieved, but the complexity and cost increase significantly
Solution Approach 1:
The first-stage multiplex PCR uses a universal set of primers that can simultaneously detect multiple different pathogens by targeting conserved genes across bacterial, viral, and fungal organisms. This single multiplex assay replaces the need for multiple separate PCR assays, reducing complexity while maintaining comprehensive detection coverage.
3Reliability
If nested secondary PCR reactions are performed to increase robustness, then amplification reliability is improved, but the risk of contamination and handling errors increases
Solution Approach 1:
The potential amplicons from the first-stage multiplex PCR are extracted and used as templates for the second-stage confirmation PCR. This extraction step allows the second-stage reaction to be performed in a separate, controlled environment, reducing contamination risk while maintaining amplification robustness through targeted confirmation of specific targets.
4Measurement precision
If sample volume is increased to gather adequate reaction templates for low concentration pathogens, then detection sensitivity is improved, but the availability of sample becomes limited
Solution Approach 1:
The first-stage multiplex PCR combines multiple amplification targets into a single reaction mixture, allowing adequate template gathering from limited sample volumes. By multiplexing the initial amplification, the system achieves sufficient sensitivity for low-concentration pathogens without requiring large sample volumes that would be needed if running separate assays.
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 enables rapid, sensitive, and robust identification of bacterial species and antibiotic resistance with reduced contamination risks, facilitating timely diagnosis and efficient use of resources.
Implementation Method 1
In recent years, the polymerase chain reaction (PCR) has become a method of choice for rapid diagnosis of infectious agents
Implementation Method 2
amplifying, in a single reaction mixture containing nucleic acid from the organism, a plurality of conserved genes using outer first-stage primers designed to hybridize to generally conserved regions of the respective genes
Data Source
AI summary
Methods and containers are provided for identifying a species, illustratively a bacterial species. Illustrative methods comprise amplifying various genes in the nucleic acid from the bacterial species in a single reaction mixture using pairs of outer first-stage primers designed to hybridize to generally conserved regions of the respective genes to generate a plurality of first-stage amplicons, dividing the reaction mixture into a plurality of second-stage reactions, each using a unique pair of second-stage primers, each pair of second-stage primers specific for a target bacterial species or subset of bacterial species, detecting which of the second-stage reactions amplified, and identifying the bacterial species based on second-stage amplification. Methods for determining antibiotic resistance are also provided, such methods also using first-stage primers for amplifying genes known to affect antibiotic resistance a plurality of the second-stage reactions wherein each pair of second-stage primers specific for a specific gene for conferring antibiotic resistance.