Multiplex Respiratory Virus PCR Detection in a Single Tube
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Solution Overview
Problem
Current methods for detecting influenza A virus, influenza B virus, and 2019-nCoV nucleic acids face challenges such as low accuracy, lengthy detection times, and difficulty in differentiating between similar clinical symptoms, with existing kits having low clinical detection accuracy and requiring multiple tubes for simultaneous detection, leading to inefficiencies.
Innovation Solution
A multiplex detection method and kit using a primer pair set and probe set in a real-time fluorescent quantitative PCR platform that allows simultaneous detection of influenza A virus, influenza B virus, and 2019-nCoV in a single reaction tube, utilizing different fluorescent channels to minimize interference and incorporating a human housekeeping gene as an internal standard to monitor sample quality.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple pathogens are detected using separate fluorescent PCR tubes, then detection specificity is maintained, but detection time increases and operational efficiency decreases
Solution Approach 1:
The patent combines detection of multiple respiratory pathogens (influenza A, influenza B, and 2019-nCoV) into a single multiplex fluorescent PCR reaction tube. Different pathogen-specific probes are labeled with distinct fluorescent dyes that emit at different wavelengths, allowing simultaneous detection of multiple targets in one reaction system without compromising specificity.
Solution Approach 2:
The invention creates a universal detection platform that can identify multiple different pathogens using a single PCR reaction system. The method employs a panel of primers and fluorescently-labeled probes that can simultaneously detect influenza A virus, influenza B virus, and 2019-nCoV, making the system multi-functional for respiratory pathogen surveillance.
2Adaptability or versatility
If high-throughput sequencing is used to detect multiple unknown sequences, then detection comprehensiveness improves, but detection cycle lengthens and operational complexity increases
Solution Approach 1:
The patent segments the detection process by designing specific primer and probe sets for each target pathogen (influenza A, influenza B, 2019-nCoV). Each pathogen is detected using dedicated fluorescent probes with unique spectral signatures, allowing comprehensive multi-pathogen detection through a structured, organized approach rather than random sequencing.
Solution Approach 2:
The invention replaces the complex mechanical and computational system of high-throughput sequencing with a simpler optical detection system. Fluorescent probes emit light signals that are detected and analyzed by standard real-time PCR instrumentation, eliminating the need for complex sequencing machinery and bioinformatics analysis while maintaining comprehensive detection capability.
3Speed
If constant temperature amplification is applied, then detection speed increases, but equipment maturity decreases and additional instruments are required
Solution Approach 1:
The patent utilizes the thermocycling capability of existing real-time PCR instruments to perform multiplex amplification and detection. The method is compatible with standard fluorescent PCR platforms, eliminating the need for specialized constant temperature amplification equipment while maintaining rapid detection capability through optimized primer and probe design.
4Measurement precision
If single pathogen detection is performed in separate tubes, then detection accuracy is maintained, but operational simplicity decreases
Solution Approach 1:
The patent merges multiple single-pathogen detection protocols into a single multiplex reaction. By combining pathogen-specific primers and fluorescent probes in one tube with optimized concentrations, the method maintains detection accuracy for each target while simplifying the workflow to a single reaction setup, incubation, and detection step.
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 high sensitivity, specificity, and simplicity in detecting these viruses with a minimum detection limit of 200 copies/mL, reducing false negatives and enabling rapid, reliable diagnosis.
Implementation Method 1
a first probe specific to the influenza A virus and labeled with a first fluorescent reporter group; a second probe specific to the influenza B virus and labeled with a second fluorescent reporter group; a third probe specific to the 2019-nCoV and labeled with a third fluorescent reporter group
Data Source
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AI summary
Provided in the present invention are a method and a kit for multiple detection of respiratory virus nucleic acids, and in particular, disclosed are a method, a primer, a probe and a kit for detecting a plurality of influenza A viruses such as H1N1(2019), H3N2, H5N1, H1N1 and H7N9, influenza B viruses such as Yamagata and Victoria, 2019-nCoV OFRlab and N genes, and human internal standard gene GAPDH on the basis of a real-time fluorescent quantitative PCR technical platform.