Isothermal LAMP Primer Set for Rapid Pathogen Detection
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Solution Overview
Problem
Current nucleic acid amplification techniques for pathogen detection in animals and plants are costly, labor-intensive, and require complex equipment and skilled personnel, with high risks of contamination and lengthy diagnostic processes, limiting their practical application in field surveys.
Innovation Solution
An integrated microfluidic LAMP system that simplifies nucleic acid extraction and purification, using specific primer sets and probes for loop-mediated isothermal amplification, enabling rapid, sensitive, and specific detection of pathogens with low sample consumption and energy usage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR-based nucleic acid amplification is used for pathogen detection, then detection sensitivity and specificity are improved, but equipment complexity and operational cost increase
Solution Approach 1:
The patent replaces the thermal cycler (mechanical temperature cycling system) with an isothermal amplification system using Bst DNA polymerase that operates at a constant temperature of 60-65°C. This substitution eliminates the need for complex temperature cycling equipment while maintaining high detection sensitivity through the LAMP method's strand displacement mechanism
Solution Approach 2:
The patent changes the temperature parameter from cyclic variation (42-95°C in PCR) to a constant isothermal condition (60-65°C in LAMP). This parameter change simplifies the equipment requirements while the four-primer system (F3, B3, FIP, BIP) maintains specific and sensitive amplification of target nucleic acid sequences
2Measurement precision
If conventional PCR-based diagnostic methods are used, then detection accuracy is improved, but diagnostic time and operational complexity increase
Solution Approach 1:
The patent implements continuous isothermal amplification without the repeated heating and cooling cycles of conventional PCR. The Bst DNA polymerase continuously synthesizes new DNA strands at constant temperature, and the strand displacement mechanism ensures continuous amplification, reducing diagnostic time from hours to minutes while maintaining accuracy through the specific primer sets
Solution Approach 2:
The patent incorporates preliminary actions by designing primers (FIP and BIP) that contain internal complementary sequences forming hairpin structures. These pre-configured primers automatically initiate strand displacement and amplification upon binding to the target, eliminating the need for complex operational steps during the diagnostic process
3Reliability
If RNA extraction and purification steps are included in the diagnostic process, then detection reliability is improved, but operational complexity and contamination risk increase
Solution Approach 1:
The patent merges the nucleic acid extraction and amplification steps into a single integrated reaction system. The LAMP reagents and buffers are designed to work directly with crude nucleic acid extracts without requiring separate purification steps, reducing operational complexity and contamination risk while maintaining detection reliability through the robustness of the isothermal amplification system
Solution Approach 2:
The patent creates a universal diagnostic system where the same LAMP reaction mixture can handle different sample types (tissue, fluid, swab) without requiring separate extraction protocols. The buffer system and primer designs are universally applicable across different pathogen types, simplifying the overall diagnostic process while maintaining reliable detection
4Productivity
If thermal cycler equipment is used for nucleic acid amplification, then amplification efficiency is improved, but cost and portability worsen
Solution Approach 1:
The patent replaces the bulky thermal cycler with a simple water bath or heating block that maintains a constant temperature of 60-65°C. This mechanical substitution dramatically reduces equipment weight and improves portability while the Bst DNA polymerase-based LAMP system maintains high amplification efficiency through its strand displacement mechanism that does not require temperature cycling
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
The system achieves high specificity and sensitivity, reducing diagnostic time to 65 minutes and minimizing human intervention, with a detection limit 1000 times higher than conventional PCR, making it suitable for early disease detection and economic protection.
Implementation Method 1
Specific nucleic acid sequences in the target test samples can be amplified by using four designated primers with the incorporation of Bst DNA polymerase, which is capable of high strand displacement under isothermal conditions (about 60° C.-65° C.)
Implementation Method 2
Bst DNA polymerase, which is capable of high strand displacement under isothermal conditions
Data Source
AI summary
The invention provides a method for rapidly detecting a pathogen in animals or plants comprising using a specific primer set and nucleic acid in a sample to carry on a loop-mediated isothermal amplification. If at least one amplification occurs, the sample comprises the pathogen. The invention also provides a primer set, probe and kit for detecting a pathogen in animals or plants.