Primer and Probe Sets for 23-Pathogen Transplant Detection
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Solution Overview
Problem
Current pathogen detection methods for transplant patients are limited by high costs, complexity, long culture cycles, low specificity, and high false positive rates, particularly in detecting multiple pathogens simultaneously.
Innovation Solution
Development of primer and probe sets specifically designed for 23 pathogens with high infection and mortality rates post-transplant, allowing simultaneous detection on a 96-well plate or TAC microfluidic chip, using real-time fluorescence quantitative PCR with labeled probes to avoid primer dimers and non-specific amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple primer pairs are added to detect multiple pathogens simultaneously, then detection coverage increases, but primer dimers and non-specific amplification occur causing high false positive rates and low specificity
Solution Approach 1:
The patent divides the detection system into separate reaction wells, with each well containing a single primer pair for one pathogen. This segmentation prevents primer-dimer formation between different primer pairs while maintaining the ability to detect multiple pathogens by running parallel reactions in a 96-well plate format.
Solution Approach 2:
The patent introduces labeled probes as intermediaries that specifically bind to target sequences and generate fluorescence signals. These probes act as mediators between the DNA amplification process and the detection system, enabling specific pathogen identification without direct interaction between multiple primer pairs.
2Reliability
If culture method is used for pathogen detection, then high specificity is achieved, but long culture cycle makes it time-consuming and labor-intensive
Solution Approach 1:
The patent replaces the mechanical culture process with a molecular biology-based PCR detection system. Instead of relying on pathogen growth in culture media over days, the method uses DNA amplification and probe hybridization to detect pathogen presence within hours, dramatically reducing detection time while maintaining specificity through labeled probes.
3Measurement precision
If SYBR Green I method is used for detection, then high sensitivity is achieved, but primer dimers and non-specific amplification result in high false positive rate and low specificity
Solution Approach 1:
The patent introduces sequence-specific labeled probes as intermediaries that must hybridize to the target sequence to generate a fluorescence signal. This intermediary step ensures that only specific target amplification produces detectable signals, eliminating false positives from non-specific amplification while retaining the sensitivity of fluorescence-based detection.
4Adaptability or versatility
If Taqman real-time fluorescence quantitative PCR method is used for combined detection, then multiple pathogens can be detected at one time with improved specificity, but cost increases
Solution Approach 1:
The patent creates a universal detection platform using a 96-well plate format where each well can detect a different pathogen using the same basic PCR and fluorescence detection methodology. This multi-functional system allows simultaneous detection of multiple pathogens using affordable reagents and standard laboratory equipment, reducing overall cost compared to specialized methods.
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 and specificity, enabling rapid and accurate detection of multiple pathogens with reduced manual intervention and operational complexity, thereby lowering mortality risks in transplant patients.
Implementation Method 1
using real-time fluorescence quantitative PCR with labeled probes
Data Source
AI summary
The present disclosure relates to primer and probe sets for pathogen detection of infection in a transplant patient, a kit and use thereof, and belongs to the technical field of molecular biology detection. There are 23 primer and probe sets that can be used to jointly detect 23 kinds of pathogens with a high infection rate and a high lethality rate after transplantation, including an adenovirus type B; and two ends of a corresponding sequence of each probe have correspondingly a fluorophore and a quencher group, respectively. The present disclosure further provides a real-time fluorescence quantitative PCR kit for pathogen detection of infection in a transplant patient, including the primer and probe sets, a pathogen plasmid standard, a fluorescence quantitative PCR reaction solution, and sterile deionized water, which can simultaneously detect 23 pathogens infected by the transplant patient.