Multiplex Pathogen Detection Composition Using Single-Channel Melting Curves
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Solution Overview
Problem
Existing methods for detecting encephalitis, meningitis, and respiratory tract pathogens are time-consuming, have low diagnostic efficiency, and struggle with sensitivity, especially in multi-pathogen infections, and existing PCR techniques are limited by the number of detection channels in a single tube.
Innovation Solution
A composition using specific primer pairs with different fluorescent reporter groups and Tm values for 15 pathogens, allowing simultaneous detection in a single channel through melting curve analysis, optimizing PCR reaction systems without altering hardware.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple PCR techniques are used to detect multiple pathogens, then clinical sensitivity increases, but device complexity and detection cost increase
Solution Approach 1:
The patent combines multiple pathogen detection capabilities into a single PCR reaction tube by using different fluorescent reporter groups (FAM, HEX, ROX, CY5) to label different target sequences. This allows simultaneous detection of multiple pathogens (including encephalitis viruses, meningitis bacteria, and respiratory tract pathogens) in one reaction, reducing the need for multiple separate PCR instruments and procedures while maintaining high clinical sensitivity
Solution Approach 2:
The invention creates a universal detection system that can identify 15 different types of pathogens using a single fluorescent quantitative PCR instrument. The system uses a standardized set of primers and fluorescent reporters that can detect various pathogens (Japanese encephalitis virus, enterovirus, herpes simplex virus, Streptococcus pneumoniae, Haemophilus influenzae, etc.) through a common detection platform, making the system multi-functional without requiring separate specialized equipment for each pathogen
2Productivity
If the number of detection channels in a single tube is increased, then throughput improves, but optimization of PCR reaction systems becomes more difficult
Solution Approach 1:
The patent applies local quality by assigning different fluorescent reporter groups to different target sequences within the same reaction tube. Each primer pair is specifically designed with a particular fluorescent label (FAM for Japanese encephalitis virus and enterovirus, HEX for herpes simplex virus, ROX for Streptococcus pneumoniae and Haemophilus influenzae, CY5 for other targets), allowing the detection system to distinguish multiple pathogens simultaneously through their unique fluorescent signatures while maintaining optimized PCR conditions for all targets
Solution Approach 2:
The invention utilizes parameter changes by detecting different fluorescent wavelengths emitted by various reporter groups. The system monitors fluorescence signals at different wavelengths (corresponding to FAM, HEX, ROX, and CY5 dyes) during the PCR amplification process, enabling simultaneous quantification of multiple pathogens in a single reaction tube without interfering with each other's amplification efficiency
3Measurement precision
If traditional detection methods are used, then diagnostic accuracy is maintained, but time consumption increases
Solution Approach 1:
The patent merges multiple traditional detection procedures into a single real-time fluorescent quantitative PCR reaction. Instead of performing separate culture, biochemical identification, and individual PCR tests for different pathogens, the system combines all detections into one automated reaction that provides results for multiple pathogens simultaneously, dramatically reducing total detection time while maintaining or improving diagnostic accuracy through real-time monitoring
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
Enables the simultaneous detection of 15 pathogens in a single tube, improving throughput and efficiency, and provides a rapid molecular diagnosis for multiple pathogens, including a kit and method for detection.
Implementation Method 1
a primer pair for Haemophilus influenzae, sequences of which are respectively as shown in SEQ ID NO: 1 and SEQ ID NO: 17; a primer pair for Streptococcus pneumoniae, sequences of which are respectively as shown in SEQ ID NO: 2 and SEQ ID NO: 18... sequences SEQ ID NO: 1 to SEQ ID NO: 16 carry fluorescent reporter groups
Implementation Method 2
carrying out a melting curve analysis method according to different Tm values of fluorescence products used by multiple targets in a single channel
Data Source
AI summary
Provided is a composition for detecting multiple encephalitis/meningitis/respiratory pathogens. The composition includes primer sequences for the pathogens as shown in SEQ ID NOs: 1-7, 9-23 and 25-32, in which SEQ ID NOs: 1-7 and 9-16 carry fluorescent reporter groups. In addition, the present invention further provides a use of the foregoing composition in the preparation of a kit, and a related kit and a method for using the same.