Real-Time PCR Nematode Identification via Probe Specificity
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Solution Overview
Problem
Current methods for identifying nematode species, such as Bursaphelenchus mucronatus, Mesocriconema xenoplax, and Rotylenchus uniformis, face challenges in sensitivity, specificity, and accuracy, particularly in soil samples and at different developmental stages, with existing genetic techniques often resulting in errors and inability to detect certain species.
Innovation Solution
A Real-Time PCR method using specifically designed 3' and 5' primers and probes, optimized for each nematode species, allows for high sensitivity and specificity in soil sample analysis, independent of sample type, origin, and developmental stage, with a reaction mixture containing buffer, Taq polymerase, magnesium ions, and primers, conducted at controlled thermal parameters.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional PCR with fluorescent dyes is used, then amplification sensitivity is improved, but reaction specificity deteriorates due to binding to all double-stranded DNA fragments
Solution Approach 1:
The patent introduces a probe as an intermediary element between the primers and the fluorescent dye. The probe specifically hybridizes to the target DNA sequence and carries the fluorescent label, ensuring that fluorescence is only generated when the probe binds to the correct target sequence. This resolves the contradiction by maintaining sensitivity (through amplification) while improving specificity (through probe-mediated detection).
Solution Approach 2:
The patent applies local quality by placing the fluorescent label specifically on the probe that binds to the target sequence, rather than using a general fluorescent dye that binds to all double-stranded DNA. This localized placement of the fluorescent marker ensures that signal generation occurs only at the specific target location, resolving the specificity issue while maintaining amplification sensitivity.
2Loss of time
If Real Time PCR with fluorescent dyes is used, then analysis time is reduced by eliminating electrophoresis, but measurement precision deteriorates due to limited reaction specificity
Solution Approach 1:
The probe acts as an intermediary that enables specific detection during Real Time PCR without requiring post-amplification electrophoresis. The probe hybridizes to the target sequence during the amplification process, and fluorescence is measured in real-time, maintaining both time efficiency and measurement precision.
3Productivity
If PCR with standard primers is used, then amplification efficiency is improved, but manufacturing precision deteriorates due to primer binding to sequences with 1-2 nucleotide mismatches
Solution Approach 1:
The patent applies local quality by designing the probe with high specificity to bind only to the exact target sequence. The probe's binding region is locally optimized to match the target sequence perfectly, ensuring that even with standard primers that may bind to sequences with 1-2 mismatches, the probe will only generate a signal when bound to the correct target sequence, thus improving manufacturing precision without sacrificing amplification efficiency.
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 rapid and accurate identification of nematodes within a few hours, significantly improving reaction specificity and speed compared to traditional PCR methods, allowing detection in soil samples regardless of developmental stage or sample characteristics.
Implementation Method 1
The principle of PCR is amplification of DNA fragment(s), specific to the given organism, up to a level allowing a rapid and simple detection thereof using electrophoresis. This is achieved by using short singlestranded oligonucleotides (12-40 nucleotides), the so called primers, specific to the amplified DNA fragment and an enzyme - a thermostable polymerase, enabling amplification of the desired fragment in a cyclic three-stage reaction, composed of denaturation, primer annealing and DNA synthesis.
Implementation Method 2
In order to measure the amplified fragment quantity fluorochromes (fluorescent dyes) are used, e.g.: SYBR Green I, SYTO9, Eva Green, SYBR Gold, fluorescence of which is proportional to the quantity of the amplified fragment.
Implementation Method 3
an enzyme - a thermostable polymerase, enabling amplification of the desired fragment in a cyclic three-stage reaction, composed of denaturation, primer annealing and DNA synthesis
Data Source
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AI summary
The object of the invention is a method and a kit for identification of nematodes, forest plant pests, by Real-Time PCR, in particular, the invention relates to a method and a kit for identification of nematodes comprising the species Bursaphelenchus mucronatus, Mesocriconema xenoplax and Rotylenchus uniformis. Due to the specifically selected primers and probes the solution according to the invention enables accurate identification of the selected nematode species regardless of the analyzed sample type, their origin and purpose, as well as developmental stage.