One-Pot Nucleic Acid Confirmation via Hapten-Labeled FEN Probes
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Solution Overview
Problem
Current nucleic acid amplification confirmation tests face challenges such as contamination risks, complexity, and limited multiplex capability, particularly in decentralized diagnostics, which can lead to inaccurate diagnoses and increased healthcare costs.
Innovation Solution
A one-pot method for amplifying and confirming target sequences using a continuous reaction mixture with hapten pair-labeled artificial template amplicons and FEN probes, which allows for simultaneous amplification and detection without additional processing steps, reducing contamination risks and enabling higher multiplexing capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If subsequent NAT confirmation steps such as hybridization of DNA amplicons on immobilized probes are performed, then confirmation of target sequences is achieved, but additional processing steps are required which are more time-consuming and expensive and carry the risk of contamination
Solution Approach 1:
The patent combines the amplification reaction and confirmation detection into a single continuous one-pot reaction. The FEN probe is included in the PCR reaction mixture from the beginning, and after amplification, the same reaction mixture is directly applied to the immunochromatographic strip without intermediate processing steps. This merging of steps eliminates contamination risks and reduces time and cost while maintaining confirmation accuracy.
Solution Approach 2:
The FEN probe is pre-labeled with hapten in the reaction mixture before amplification occurs. This preliminary labeling ensures that when the probe hybridizes to the target sequence during or after amplification, the labeled amplicons are already prepared for immediate detection on the immunochromatographic strip, eliminating the need for subsequent labeling steps.
2Productivity
If homogeneous test procedures with fluorescent dyes are used, then real-time detection is achieved, but multiplexing capability is limited to 3-6 different detection channels
Solution Approach 1:
The patent uses haptens as intermediary molecules that can be detected by immunochromatographic methods. Multiple different haptens can be incorporated into different FEN probes, allowing simultaneous detection of multiple target sequences. The immunochromatographic strip contains corresponding receptor molecules for each hapten, enabling high-capacity multiplexing beyond the limitations of fluorescent channel detection.
3Reliability
If aliquots are taken from the PCR reaction and transferred to a new reaction for confirmatory testing, then confirmation can be performed, but contamination with foreign DNA or amplicons from parallel-processed samples occurs
Solution Approach 1:
The patent merges the confirmation detection step with the amplification reaction by using the same reaction mixture for both purposes. The labeled FEN probe remains in the PCR tube throughout amplification, and the entire reaction mixture is directly applied to the immunochromatographic strip. This eliminates the need to transfer aliquots between reactions, thereby preventing cross-contamination while maintaining confirmation accuracy.
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 simplifies the confirmation process, reduces contamination risks, and enhances sensitivity, enabling accurate diagnostics even with low DNA quantities, while allowing for decentralized and cost-effective point-of-care testing.
Implementation Method 1
The 5' cleavage product of the at least one target sequence-specific FEN probe is obtained only if the FEN probe hybridizes with its target sequence-specific 3' sequence to a complementary sequence segment of the at least one target sequence
Implementation Method 2
which, after hybridization, is activated by the intrinsic nuclease activity of the Taq DNA polymerase cleaves the DNA, separating the fluorophore pair
Implementation Method 3
The detection of the resulting plurality of template amplicons is performed distinctly and preferably by immunochromatographic methods
Data Source
Figure 1A~2B
Figure 3A~4
Figure 5
AI summary
The invention relates to a method for confirming an amplified nucleic acid target sequence (target sequence), preferably from human samples, during a multiplication reaction in a collective and continuous reaction batch as a one-pot process, wherein the confirmation of the target sequence amplification product is obtained by means of a hapten-pair-marked artificial template amplification product. The artificial template sequence is amplified and optionally marked by means of the 5' cleavage products of the at least one target-sequence-specific FEN probe. The 5' cleavage product of the at least one target-sequence-specific FEN probe is obtained only if the FEN probe hybridizes, by means of the target-sequence-specific 3' sequence thereof, to a complementary sequence segment of the at least one target sequence. The detection of the obtained plurality of template amplification products occurs distinctly and preferably by means of immunochromatographic methods.