Biphasic DNA Amplification with Solid-Phase Target Capture
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Solution Overview
Problem
Current methods for detecting pathogens in biological samples, such as PCR, suffer from low sensitivity due to interference from non-target nucleic acids, leading to false negatives or positives, and are costly and time-consuming for identifying polymorphisms in antibiotic resistance genes.
Innovation Solution
A biphasic amplification reaction combining solid and solution-phase amplification using immobilized and non-immobilized primers, with real-time detection via ISFETs or optical imaging, allowing for sequence-specific amplification and detection of nucleic acid fragments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional PCR methods are used to detect pathogen nucleic acids, then amplification of target sequences can be achieved, but sensitivity is reduced due to interference from non-target human nucleic acids
Solution Approach 1:
The invention divides the amplification process into two distinct phases: a solid phase where target nucleic acids are captured and immobilized on a surface, and a solution phase where amplification occurs in solution. This segmentation allows selective enrichment of target sequences while separating them from inhibitory non-target nucleic acids, thereby improving detection sensitivity
Solution Approach 2:
The invention introduces a solid support surface as an intermediary between the sample and the amplification reagents. This intermediary enables selective capture and concentration of target nucleic acids through hybridization with immobilized capture probes, while excluding non-target nucleic acids that would otherwise interfere with the amplification reaction
2Adaptability or versatility
If multiple primer pairs are combined for multiplex PCR to detect different sequences, then amplification capability is improved, but cross-hybridisation between primers occurs leading to non-specific amplification
Solution Approach 1:
The invention segments the primer functions by separating capture probes (immobilized on solid support) from amplification primers (in solution). This allows multiple capture probes targeting different sequences to be immobilized on the same solid support without cross-hybridization, while amplification primers are added in solution for each target, enabling true multiplexing without cross-reactivity
Solution Approach 2:
The invention transitions from a conventional solution-phase multiplex PCR to a biphasic system where capture specificity is established in the solid phase and amplification occurs in the solution phase. This dimensional separation allows multiple specific interactions to occur simultaneously on the solid support surface without interfering with each other in solution
3Reliability
If blood culture methods are used for pathogen identification, then definitive diagnosis can be obtained, but the process is costly and time-consuming
Solution Approach 1:
The invention performs preliminary capture and concentration of target nucleic acids on a solid support before amplification. This preliminary enrichment step increases the starting template concentration and specificity, allowing rapid amplification and detection within hours rather than days, while maintaining diagnostic accuracy comparable to culture methods
Solution Approach 2:
The invention replaces the biological culture system with a nucleic acid-based detection system. Instead of relying on slow bacterial growth in culture media, the method directly detects and amplifies pathogen nucleic acids using the biphasic PCR approach, dramatically reducing turnaround time while preserving diagnostic 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
Enhances the sensitivity and specificity of nucleic acid amplification, reducing false results and enabling rapid, cost-effective identification of target sequences, including polymorphisms, by using a combination of immobilized and non-immobilized primers and real-time detection.
Implementation Method 1
a solid support having one or more immobilised amplification primers
Implementation Method 2
The pH can be monitored in real time during the amplification reaction using an ISFET sensor
Data Source
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AI summary
Described is a means for efficiently amplifying and detecting certain nucleic acid sequences from a population. The selected population can be further characterised, for example by sequencing. The method involves combined solution phase and solid phase amplification.