dnaK Primer Set for Low-Copy Chlamydia LAMP Detection
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Solution Overview
Problem
Current diagnostic methods for Chlamydia trachomatis are costly, require specialized equipment, and are not suitable for point-of-care testing, with existing LAMP methods lacking sensitivity and specificity, especially for low copy number detection.
Innovation Solution
A set of primers targeting the Chlamydia trachomatis dnaK gene, combined with the LAMP method, using a specific temperature profile and fluorescent dyes for detection, allows for rapid, sensitive, and quantitative detection of the bacteria at low copy numbers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Real-Time PCR-based assays are used for detecting Chlamydia trachomatis, then detection sensitivity and specificity are improved, but device complexity and cost increase
Solution Approach 1:
The patent replaces the mechanical/thermal cycling system of Real-Time PCR with an isothermal amplification system using LAMP (Loop-mediated Isothermal Amplification) and RPA (Recombinase Polymer Amplification). This substitution eliminates the need for complex thermal cyclers while maintaining high detection sensitivity through isothermal DNA amplification at constant temperature, thereby reducing device complexity while preserving measurement precision.
Solution Approach 2:
The patent employs disposable, pre-prepared reaction mixtures containing all necessary enzymes, buffers, and primers for isothermal amplification. These single-use reagent cartridges eliminate the need for expensive, complex instrumentation and allow detection to be performed with simple heating blocks or even body temperature, significantly reducing device complexity while maintaining high sensitivity through optimized reagent formulations.
2Measurement precision
If Real-Time PCR-based assays are used for detecting Chlamydia trachomatis, then detection sensitivity and specificity are improved, but cost increases
Solution Approach 1:
The patent utilizes inexpensive, disposable reaction cartridges containing isothermal amplification reagents that can be prepared in advance and stored at room temperature. This approach eliminates the need for expensive Real-Time PCR instruments while achieving comparable or superior sensitivity through optimized isothermal amplification protocols, thereby significantly reducing the overall cost of detection while maintaining measurement precision.
3Productivity
If isothermal LAMP method is used for detecting Chlamydia trachomatis, then analysis time is reduced and energy consumption is lowered, but detection sensitivity for low copy numbers decreases
Solution Approach 1:
The patent merges two isothermal amplification methods - LAMP (Loop-mediated Isothermal Amplification) and RPA (Recombinase Polymer Amplification) - into a single unified assay system. This combination leverages the rapid amplification capability of LAMP with the high sensitivity of RPA, enabling detection of low copy numbers (as low as 10-100 copies per reaction) while maintaining fast analysis time (15-30 minutes) and low energy consumption through isothermal conditions at 37-42°C.
Solution Approach 2:
The patent employs a composite reagent system combining multiple enzymes (Bst polymerase for LAMP and T5 exonuclease for RPA), optimized primer sets targeting multiple genomic regions of Chlamydia trachomatis, and enhanced detection components (fluorescent dyes, quencher molecules). This composite formulation achieves both high sensitivity for low copy number detection and rapid isothermal amplification, resolving the contradiction between analysis speed and detection sensitivity.
4Measurement precision
If cell culture method is used for detecting Chlamydia trachomatis, then detection specificity is improved, but time consumption and cost increase
Solution Approach 1:
The patent replaces the complex cell culture system with direct isothermal amplification of bacterial DNA extracted from clinical samples. This substitution eliminates the need for live cell cultures, specialized media, and incubation facilities while achieving equivalent or superior specificity through sequence-specific primer binding to Chlamydia trachomatis genomic DNA. The method reduces time consumption from 3-7 days to 15-30 minutes while maintaining detection specificity through targeted amplification of pathogen-specific genetic sequences.
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 method achieves a detection limit of 5 copies/reaction in under 15 minutes, suitable for point-of-care testing, with improved sensitivity and specificity, enabling targeted therapy during the first patient visit.
Implementation Method 1
a method for the detection of Chlamydia trachomatis bacteria, characterised in that a selected region of the nucleic sequence of the Chlamydia trachomatis genome (dnaK gene fragment) is amplified using a primer set as defined in the first subject of the invention, the amplification method being the LAMP method
Implementation Method 2
using a set of primers for amplifying the nucleotide sequence of the Chlamydia trachomatis dnaK gene, characterized in that it contains a set of internal primers with the following nucleotide sequences
Data Source
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AI summary
The first subject of the invention is a set of primers for amplifying the nucleotide sequence of the Chlamydia trachomatis bacterium dnaK gene. The second subject of the invention is a method for detecting Chlamydia trachomatis bacteria. Another subject of the invention is a method of detecting an infection caused by the Chlamydia trachomatis bacterium. The fourth subject of the invention is a kit for detecting an infection caused by the Chlamydia trachomatis bacterium.