LAMP Primer Loop Design for HPV Genotype Detection
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Solution Overview
Problem
Current methods for detecting human papillomavirus (HPV) are either complex, require expensive enzymes or magnetic beads, or suffer from low hybridization efficiency due to double-stranded DNA products competing with probes in PCR-based detection, making them unsuitable for rapid and accurate genotype identification.
Innovation Solution
The use of a nucleic acid primer designed for LAMP amplification, where the HPV target sequence is placed in a single-stranded loop region, allowing for specific hybridization without denaturation, and a nucleic acid probe immobilized on a support for efficient detection of HPV genotypes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If PCR method is used for HPV detection, then amplification of target sequence is achieved, but the procedure becomes complex and requires expensive equipment
Solution Approach 1:
The patent replaces the complex thermal cycling mechanical system of PCR with an isothermal LAMP amplification system that uses a simple water bath or heating block, eliminating the need for expensive thermal cyclers while maintaining amplification reliability
Solution Approach 2:
The patent extracts and eliminates the complex pretreatment steps (nucleic acid extraction, purification) from the detection workflow by using a one-step LAMP assay that can be performed directly on clinical samples, significantly simplifying the overall procedure
2Reliability
If PCR amplification is used, then target DNA is amplified, but reaction time extends to two hours or more
Solution Approach 1:
The patent changes the amplification parameters from thermal cycling conditions to isothermal conditions at 65°C, enabling rapid LAMP amplification that completes in 15-30 minutes while maintaining high amplification efficiency and reliability
Solution Approach 2:
The patent uses periodic addition of primers during the isothermal amplification process to maintain high amplification efficiency throughout the reaction, achieving rapid and reliable detection within 15-30 minutes
3Productivity
If double-stranded PCR products are used for hybridization, then amplification is complete, but hybridization efficiency decreases due to complementary strand competition
Solution Approach 1:
The patent inverts the conventional approach by designing LAMP primers that generate single-stranded DNA products with hairpin structures, where the target sequence is exposed in the loop region rather than being sequestered in double-stranded regions, thereby eliminating complementary strand competition and improving hybridization efficiency
Solution Approach 2:
The patent creates local single-stranded regions in the LAMP product structure where the target sequence is exposed and accessible for probe hybridization, while other regions form stable hairpin structures, optimizing both amplification completeness and hybridization efficiency
4Device complexity
If DNA/RNA-recognizing antibody method is used, then detection is simplified, but genotype identification capability is lost
Solution Approach 1:
The patent creates a universal LAMP assay platform that can detect and identify multiple HPV genotypes using the same simplified isothermal amplification method, combining the simplicity of antibody-based detection with the genotype identification capability of molecular methods through probe hybridization
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 simple, rapid, and accurate detection of HPV genotypes with improved hybridization efficiency and reduced costs, capable of identifying polymorphisms and mutations in HPV sequences.
Implementation Method 1
the primer is so designed that the human papilloma virus-derived target sequence becomes located in the single-stranded loop region of the LAMP product... allowing for specific hybridization without denaturation
Implementation Method 2
employing a method different from the PCR method, i.e., LAMP method, for identification of the HPV genotype
Data Source
AI summary
Provided is a nucleic acid primer for LAMP amplification for use in the detection of human papilloma virus and identification of its genotype. The present invention also provides a method of detecting human papilloma virus and identifying its genotype, includes a step of amplifying the nucleic acid chains in a sample in LAMP reaction by using multiple primers including at least one primer selected from the nucleic acid primers according to the present invention and a step of detecting presence of amplified products after the amplification reaction and identifying their genotypes.