dnaE LAMP Primer Set for Rapid Mycoplasma Pneumoniae Detection
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Solution Overview
Problem
Current diagnostic methods for Mycoplasma pneumoniae infections are not suitable for point-of-care settings, lack sensitivity, and require extended time, making them inefficient for rapid detection and targeted therapy.
Innovation Solution
A primer set targeting the Mycoplasma pneumoniae dnaE gene using LAMP technology, combined with a fluorescent dye, allows for rapid amplification and detection of as few as 5 copies/μl within 15 minutes, enabling quantitative measurement and storage at room temperature.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If classical microbiological methods are used to detect Mycoplasma pneumoniae, then the detection process is simple, but the method requires specific growth conditions and specialized media, making it difficult to detect bacteria in biological material
Solution Approach 1:
The patent replaces classical microbiological methods (requiring cell lines or specialized media) with molecular testing based on LAMP amplification. This substitution eliminates the need for complex growth conditions and specialized media, enabling direct detection of bacterial DNA from biological materials using standard laboratory equipment.
Solution Approach 2:
The patent changes the detection parameter from bacterial growth detection to DNA amplification detection. By detecting specific fragments of the Mycoplasma pneumoniae bacterial genome through LAMP amplification, the method transforms the detection approach to work with standard laboratory materials and equipment while maintaining high sensitivity.
2Ease of operation
If serological methods are used to detect Mycoplasma pneumoniae infection, then the detection process is straightforward, but the methods are characterized by low sensitivity, generating false-negative results due to serological window or chronic phase infection
Solution Approach 1:
The patent substitutes serological methods (detecting antibodies) with molecular testing (detecting bacterial DNA). This replacement eliminates the serological window period and false-negative results associated with antibody detection, providing immediate and accurate detection of the pathogen itself.
Solution Approach 2:
The patent performs preliminary detection of bacterial DNA before antibody production occurs. By detecting the pathogen's genetic material directly in the sample, the method enables early diagnosis before the serological window closes, avoiding false-negative results.
3Reliability
If existing LAMP methods are used to detect Mycoplasma pneumoniae, then the amplification reaction can be performed, but the time required for amplification reaction is 30 to 90 minutes, which is not suitable for rapid point-of-care diagnostics
Solution Approach 1:
The patent optimizes the LAMP reaction parameters by using a nested primer set (internal primers with fluorescent tags and external primers) that enhances amplification efficiency. This parameter optimization reduces the reaction time from 30-90 minutes to under 15 minutes while maintaining reliable detection.
Solution Approach 2:
The patent employs continuous monitoring of the amplification reaction through fluorescent signal detection. The real-time monitoring allows for earlier termination of the reaction once sufficient amplification is achieved, reducing the total time required while ensuring reliable detection results.
4Productivity
If existing LAMP methods are used, then detection can be performed, but the minimum concentration of M. pneumoniae DNA that can be detected is 1.5×10^-7 ng/μL, which limits the detection sensitivity for low bacterial loads
Solution Approach 1:
The patent uses the LAMP amplification process to create multiple copies of the target DNA sequence. The nested primer set generates exponential amplification, converting low concentrations of original DNA (as low as 5 copies/μl) into detectable amounts of amplified product, thereby enhancing detection sensitivity.
Solution Approach 2:
The patent optimizes the detection parameter by using fluorescent tagging of internal primers, which allows for sensitive detection of amplified products. This parameter change enables detection of lower DNA concentrations than existing methods while maintaining rapid detection speed.
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 primer set achieves high sensitivity and specificity for Mycoplasma pneumoniae detection, suitable for point-of-care diagnostics, with reduced analysis time and improved detection limits, facilitating timely targeted therapy.
Implementation Method 1
A primer set targeting the Mycoplasma pneumoniae dnaE gene using LAMP technology, combined with a fluorescent dye, allows for rapid amplification and detection of as few as 5 copies/μl within 15 minutes
Implementation Method 2
combined with a fluorescent dye, allows for rapid amplification and detection of as few as 5 copies/μl within 15 minutes
Data Source
AI summary
The first subject of the invention is a set of primers for amplifying nucleotide sequence of the Mycoplasma pneumoniae dnaE gene. The second subject of the invention is a method for detecting Mycoplasma pneumoniae bacteria. Another subject of the invention is a method for detecting an infection caused by the Mycoplasma pneumoniae bacterium. A fourth subject of the invention is a kit for detecting an infection caused by the Mycoplasma pneumoniae bacterium.