Double Stem-Loop Oligonucleotide LAMP Detection
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Solution Overview
Problem
Current methods for detecting molecules of interest in liquid samples, such as ELISA and PCR-based techniques, face challenges with low concentration detection, complexity, and high costs due to the need for multiple antibodies or enzymes, and require specific probe designs for each analyte.
Innovation Solution
A method using an oligonucleotide with a double stem-loop structure and LAMP amplification with two primers, which allows for sensitive detection and quantification without the need for multiple antibodies or enzymes, and can detect various analytes with improved specificity and reduced complexity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If ELISA technique with antibodies or aptamers is used, then detection can be performed with simple procedure, but detection limit is insufficient for very low concentrations
Solution Approach 1:
The patent changes the concentration parameter by introducing a signal amplification mechanism. The oligonucleotide probe bound to the analyte serves as a template for LAMP amplification, generating numerous copies of the target sequence. This exponential amplification transforms the undetectably low concentration of analyte-bound probes into a high-concentration amplification product that can be easily detected, thereby improving measurement precision without increasing procedural complexity
Solution Approach 2:
The patent introduces an intermediary oligonucleotide probe that binds to the analyte and serves as a template for LAMP amplification. This probe acts as a mediator between the analyte and the detection system, converting the binding event into an amplifiable DNA sequence. The intermediary enables the system to achieve high detection sensitivity by leveraging the exponential amplification capability of LAMP without requiring complex antibody-enzyme conjugates
2Measurement precision
If PCR-based methods are used to amplify DNA probe, then detection sensitivity is improved, but the process requires long temperature cycles and becomes more complex
Solution Approach 1:
The patent changes the temperature parameter by replacing the cyclic temperature protocol of PCR with a constant temperature LAMP amplification process. LAMP operates at a single temperature (typically 60-65°C), eliminating the need for repeated heating and cooling cycles. This parameter change maintains high detection sensitivity through exponential amplification while significantly simplifying the thermal processing requirements and reducing equipment complexity
Solution Approach 2:
The patent substitutes the mechanical temperature cycling system of PCR with the isothermal chemical amplification system of LAMP. Instead of using a thermal cycler to repeatedly heat and cool the reaction mixture, the system uses a single-temperature incubator or even a simple water bath. The amplification is driven by the biochemical properties of the Bst polymerase and the LAMP primer design, replacing the mechanical temperature modulation with a chemically-driven isothermal process
3Temperature
If LAMP amplification with four primers is used, then isothermal amplification is achieved, but primer design becomes complex and expensive
Solution Approach 1:
The patent extracts and utilizes only the essential amplification function from the four-primer LAMP system by adapting it to work with the oligonucleotide probe itself. The probe's inherent sequence structure is leveraged to function as the amplification template, eliminating the need for separate four-primer sets. This extraction simplifies the system while maintaining isothermal amplification capability, reducing primer design complexity and cost
4Measurement precision
If specific probe designs are created for each analyte, then detection specificity is improved, but the cost and time for probe preparation increases
Solution Approach 1:
The patent introduces a universal LAMP amplification system that can amplify any oligonucleotide sequence that serves as a template. Once the analyte-specific oligonucleotide probe is designed and bound to the target, the same LAMP reagents and conditions can be used for amplification regardless of the specific analyte. This universality means that while probe design remains specific to each analyte, the amplification and detection infrastructure can be standardized, reducing overall cost and preparation time across multiple analyte 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 faster, less complex, and cost-effective detection and quantification of analytes in liquid samples, with improved sensitivity and specificity, and can handle a range of analytes without the need for extensive probe customization.
Implementation Method 1
at least one oligonucleotide with a double stem-loop structure, capable of binding said analyte
Implementation Method 2
bring said surface into contact with two loop-mediated isothermal amplification primers under conditions permitting amplification of said oligonucleotide
Data Source
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AI summary
The present invention relates to a method for detecting and possibly quantifying an analyte that may be present in a liquid sample, said method employing a double stem-loop oligonucleotide and a two-primer LAMP amplification. The present invention also relates to specific double stem-loop oligonucleotides.