Real-Time PCR Primer Pair for Ehrlichia canis Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for diagnosing Ehrlichia canis infection, such as blood smears and serologic tests, are time-consuming, unreliable, and lack sensitivity, while molecular diagnosis by PCR is labor-intensive and requires end-point gel electrophoresis, making rapid and accurate detection challenging.
Innovation Solution
A primer pair and kit for real-time PCR using specific forward and reverse primers (5′-ATTAATGTACTATGCTCCAAG-3′ and 5′-GTTGAGTTTCTTTCTTCTTTAG-3′) and a probe (5′-ACTATACAAGACGATAACACTGGTAGC-3′) labeled with a reporter dye and quencher, designed for high sensitivity and specificity to detect Ehrlichia canis DNA, allowing for rapid and accurate diagnosis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If end-point PCR detection method with gel electrophoresis is used, then sensitivity and specificity are improved, but time consumption and labor intensity increase
Solution Approach 1:
The patent replaces the mechanical gel electrophoresis separation system with a fluorescent probe-based detection system. The probe labeled with reporter and quencher molecules detects amplified DNA through fluorescence signal changes, eliminating the need for gel electrophoresis equipment and procedures while maintaining high detection sensitivity and specificity.
Solution Approach 2:
The patent introduces a fluorescent probe as an intermediary between the PCR amplification process and the detection system. The probe hybridizes to the amplified DNA target and generates a fluorescent signal that can be detected in real-time, serving as a mediator that translates molecular recognition into a measurable signal without requiring post-PCR gel electrophoresis.
2Loss of time
If real-time PCR with fluorescent probe is used, then time consumption is reduced, but detection complexity increases
Solution Approach 1:
The patent merges the amplification and detection functions into a single real-time PCR process. The fluorescent probe is added to the PCR reaction mixture, and both amplification and detection occur simultaneously in the same reaction vessel, eliminating the need for separate post-PCR processing steps and reducing overall detection time.
Solution Approach 2:
The fluorescent probe system is self-detecting during the PCR amplification process. As DNA amplification occurs, the probe hybridizes to the amplified product and generates a fluorescent signal automatically, without requiring external intervention or additional processing steps. The system monitors its own progress in real-time.
3Ease of manufacture
If conventional PCR with gel electrophoresis is used, then equipment requirements are reduced, but labor intensity and time consumption increase
Solution Approach 1:
The patent replaces the mechanical gel electrophoresis system with an optical detection system based on fluorescent probes. This substitution eliminates the need for gel casting, electrophoresis running, and gel imaging procedures, significantly reducing labor intensity while maintaining diagnostic capability.
Solution Approach 2:
The real-time PCR system with fluorescent probe enables continuous monitoring of the amplification process throughout the reaction. The fluorescent signal increases continuously as DNA amplification progresses, providing real-time information without interruption, unlike the discontinuous steps of conventional PCR followed by gel electrophoresis.
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 enables high sensitivity and specificity for detecting Ehrlichia canis, facilitating early-stage diagnosis and differentiation from other tick-borne pathogens, reducing the risk of misdiagnosis and improving treatment outcomes.
Implementation Method 1
a forward primer having a sequence of 5'-ATTAATGTACTATGCTCCAAG-3' (SEQ ID NO: 1) and a reverse primer having a sequence of 5'-GTTGAGTTTCTTTCTTCTTTAG-3' (SEQ ID NO: 2). The forward primer and the reverse primer are used for real-time polymerase chain reaction.
Implementation Method 2
a probe having a sequence of 5'-ACTATACAAGACGATAACACTGGTAGC-3' (SEQ ID NO: 3). The forward primer, the reverse primer and the probe are used for real-time polymerase chain reaction. The probe is labeled with a 5'-reporter dye and a 3'-quencher.
Implementation Method 3
The probe is labeled with a 5'-reporter dye and a 3'-quencher.
Data Source
AI summary
Primer pair, kit and method for detecting Ehrlichia canis are disclosed. The primer pair includes a forward primer and a reverse primer, and the kit includes the primer pair and a probe. The forward primer has a sequence of SEQ ID NO: 1, the reverse primer has a sequence of SEQ ID NO: 2, and the probe has a sequence of SEQ ID NO: 3.