qPCR Primer-Probe Kit for Fast, Accurate Mycoplasma Detection
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Solution Overview
Problem
Current nucleic acid detection kits for Mycoplasma pneumoniae have high false positive/negative rates and require prolonged reaction times, affecting treatment accuracy and timing.
Innovation Solution
A kit comprising specific primers and probes (SEQ ID NO: 2-9) for real-time qPCR with a reporter-quencher system, enabling fast and specific detection of Mycoplasma pneumoniae in less than 1 hour, with a limit of detection as low as 5 copies per reaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional nucleic acid detection kits are used, then detection can be performed, but the reaction time is prolonged (about an hour or longer) and false positive/negative rates occur
Solution Approach 1:
The patent optimizes PCR reaction parameters including temperature profiles (denaturation at 95°C for 30s, annealing at 55-65°C for 30s, extension at 72°C for 30s), Mg2+ concentration (1.5-2.5 mM), and primer/probe concentrations to achieve faster amplification in 35-45 cycles within 1 hour while maintaining detection accuracy through precise parameter control
Solution Approach 2:
The patent uses fluorescent probe copying where the probe sequence hybridizes to the target DNA and generates a fluorescent signal copy that can be detected in real-time, allowing multiple detection events during the amplification process rather than a single end-point measurement, thereby improving accuracy without extending total time
2Reliability
If conventional detection methods are used, then detection is possible, but false positives or false negatives occur with reduced reliability
Solution Approach 1:
The patent introduces a fluorescent probe as an intermediary molecule that specifically hybridizes to the target sequence between the primers. This probe acts as a mediator that generates a fluorescent signal only when bound to the correct target, providing an additional layer of specificity verification that reduces false positives while maintaining sensitivity for true positives
Solution Approach 2:
The patent replaces conventional end-point detection methods with real-time fluorescent detection during PCR amplification. This substitution allows continuous monitoring of the amplification process, enabling detection of the exponential growth phase and providing more reliable quantification and presence/absence determination with reduced false results
3Productivity
If rapid detection is implemented, then reaction time is reduced, but detection sensitivity and accuracy may be compromised
Solution Approach 1:
The patent implements continuous fluorescent signal monitoring throughout the entire PCR amplification process rather than intermittent or end-point measurement. This continuous detection captures the exponential amplification phase in real-time, providing sufficient sensitivity even with reduced cycle numbers (35-45 cycles), and allows accurate determination of target presence/absence and quantification within the compressed 1-hour timeframe
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 kit achieves over 99% specificity, detects Mycoplasma pneumoniae with high sensitivity despite human genome interference, and reduces costs through freeze-dried reagents.
Implementation Method 1
detecting a fluorescent signal after each amplification cycle
Implementation Method 2
the 5′ end of the probe is connected to a reporter, and the 3′ end of the probe is connected to a quencher
Implementation Method 3
amplifying nucleic acids in the biological sample by performing a polymerase chain reaction
Implementation Method 4
performing a polymerase chain reaction using the reaction solution to obtain an amplification product
Implementation Method 5
the combination of primers and probe is in a form of a freeze-dried reagent
Data Source
AI summary
A kit of detecting Mycoplasma pneumoniae is provided, and the kit comprises a combination of primers and probe consisting of a forward primer, a reverse primer and a probe. The forward primer is selected from the group consisting of SEQ ID NO: 2-3 and complementary sequences thereof, the reverse primer is selected from the group consisting of SEQ ID NO: 6-8 and complementary sequences thereof, and the probe is SEQ ID NO: 9 or a complementary sequence thereof. A method for detecting Mycoplasma pneumoniae using the above kit is also provided.