IsoPCR Nucleic Acid Detection Method
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Solution Overview
Problem
Current nucleic acid amplification techniques, such as PCR, face challenges in detecting multiple target sequences simultaneously due to limited sensitivity, time-consuming processes, and the need for thermal cycling, which is problematic in point-of-care settings, especially for diagnosing conditions like sepsis where low copy numbers of nucleic acid sequences are present.
Innovation Solution
A method combining PCR pre-amplification with loop-mediated isothermal amplification, termed 'isoPCR', where a sample is pre-amplified using PCR and then subjected to isothermal amplification using specific primers, allowing for the simultaneous detection of multiple target sequences in a single reaction vessel.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR is used to amplify target nucleic acid sequences, then amplification sensitivity is improved, but the requirement for thermal cycling makes the process time-consuming and difficult to handle
Solution Approach 1:
The invention divides the amplification process into two distinct stages: a PCR pre-amplification stage for initial target enrichment, and an isothermal LAMP amplification stage for rapid exponential amplification. This segmentation allows each stage to optimize for its specific function, resolving the contradiction between sensitivity (achieved through PCR) and time efficiency (achieved through isothermal LAMP).
Solution Approach 2:
The PCR pre-amplification step serves as a preliminary action that enriches the target nucleic acid sequences before the main isothermal amplification process. By performing this enrichment step first, the subsequent isothermal amplification can proceed more efficiently with sufficient target material, reducing the overall time required while maintaining sensitivity.
2Adaptability or versatility
If multiplex PCR is used to detect multiple target sequences, then detection capability is improved, but amplification sensitivity is limited due to variability of amplification efficiencies
Solution Approach 1:
The invention separates the detection of multiple targets into distinct isothermal amplification reactions, each optimized for its specific target sequence. By avoiding simultaneous multiplex amplification and instead using sequential or parallel isothermal reactions, each target can be amplified with optimal efficiency, maintaining high sensitivity across all detected targets.
Solution Approach 2:
The invention changes the fundamental parameter of amplification conditions from thermal cycling (PCR) to isothermal conditions (LAMP). This parameter change enables more consistent and predictable amplification efficiencies across different target sequences, as isothermal amplification is less susceptible to the variability that plagues multiplex PCR.
3Adaptability or versatility
If parallel simplex PCR is used to detect multiple targets, then detection capability is improved, but the limited quantity of target DNA makes the process infeasible
Solution Approach 1:
The invention combines multiple target detections into a single isothermal amplification reaction vessel by using partitioned microreactors or spatially separated amplification zones within one container. This merging approach allows simultaneous detection of multiple targets while using the same limited sample volume, as the initial PCR pre-amplification pool serves all subsequent isothermal reactions.
Solution Approach 2:
The PCR pre-amplification step creates a pooled enriched sample that contains sufficient target material for multiple subsequent isothermal amplifications. This preliminary enrichment action ensures that even though the original sample has limited DNA quantity, there is enough amplified material to distribute across multiple detection reactions.
4Ease of operation
If isothermal amplification techniques are used to simplify the process, then ease of operation is improved, but amplification sensitivity is insufficient for very low copy numbers
Solution Approach 1:
The invention segments the amplification process into two stages with different sensitivity characteristics: PCR pre-amplification for maximum sensitivity with low copy numbers, followed by isothermal LAMP for ease of operation. This segmentation allows the system to achieve both high sensitivity and operational simplicity by letting each stage perform its strength.
Solution Approach 2:
The PCR pre-amplification serves as a preliminary sensitivity-enhancing action that boosts very low copy number targets to detectable levels before the isothermal amplification begins. This preliminary action ensures that even isothermal techniques, which normally have lower sensitivity, can work effectively with the pre-enriched target material.
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 enhances sensitivity and speed, enabling the detection of very low copy numbers of nucleic acid sequences, including multiple targets, and simplifies the process by eliminating the need for thermal cycling, making it suitable for point-of-care diagnostics.
Implementation Method 1
the requirement of a multitude of repeating cycles of thermal cycling (cycles of temperature shifts where different steps in the PCR process are taking place)
Implementation Method 2
loop-mediated isothermal amplification of DNA (LAMP)
Data Source
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AI summary
The present invention relates to a method and a kit of parts for detecting the presence or absence of one or more target nucleic acid sequences in a sample, the method comprising a sequence of steps for pre-amplifying the sample by means of a polymerase chain reaction, followed by a sequence of steps comprising an isothermal amplification of the pre-amplified sample, wherein the isothermal amplification comprises a pair of primers comprising a forward primer having a 3' part that is substantially complementary to a first part of the target sequence, the presence or absence of which is to be detected, and a 5' part that is substantially homolog to a second part of the target sequence, and a reverse primer comprising a 3' part that is substantially homolog to a fourth part of the target sequence and a 5' part that is substantially complementary to a third part of the target sequence.