qPCR Detection of Grapevine Powdery Mildew DNA
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Solution Overview
Problem
Current methods for detecting and managing powdery mildew in grapevines are hindered by the difficulty in culturing and maintaining the fungus Erysiphe necator in vitro, leading to limited molecular characterization and subjective visual assessment of infection, which complicates early detection and fungicide treatment planning.
Innovation Solution
A quantitative Polymerase Chain Reaction (qPCR) method using specific oligonucleotide primers and probes targeting the nuclear ribosomal Internal Transcribed Spacers (ITS) of Erysiphe necator, enabling sensitive and specific detection and quantification of the fungus before visible symptoms appear.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If visual assessment is used for powdery mildew detection, then the method is simple and requires no special equipment, but the detection precision is low and subjective, particularly when infection is slight
Solution Approach 1:
The patent replaces the mechanical/visual assessment system with a molecular biological system (qPCR). Instead of relying on human visual inspection of symptoms, the invention uses quantitative polymerase chain reaction to detect and quantify Erysiphe necator DNA in plant tissue, providing objective and precise measurement of fungal presence and load.
Solution Approach 2:
The patent introduces DNA as an intermediary substance for detection. Rather than directly observing fungal structures or symptoms, the method detects fungal DNA extracted from plant tissue as an intermediate step, which then allows for precise quantification of fungal presence through qPCR amplification and fluorescence measurement.
2Reliability
If qPCR method is used for early detection of Erysiphe necator, then the detection sensitivity and precision are improved, but the device complexity and cost increase
Solution Approach 1:
The patent performs preliminary DNA extraction and purification from plant tissue before the qPCR amplification step. This preliminary action isolates the target fungal DNA from complex plant tissue matrices, removing inhibitors and concentrating the target, which enables the subsequent qPCR to proceed with high sensitivity and reliability even at early infection stages.
Solution Approach 2:
The patent utilizes parameter changes in the qPCR process, specifically the exponential amplification of DNA targets through cyclic temperature changes and the corresponding fluorescence signal increase. This parameter change (from low initial DNA concentration to high amplified concentration with measurable fluorescence) enables detection of extremely low fungal loads that would be undetectable by other methods.
3Loss of information
If molecular characterization is performed on Erysiphe necator, then the genetic database information and detection accuracy improve, but the difficulty of culturing and maintaining the fungus in vitro limits the available data
Solution Approach 1:
The patent extracts DNA directly from plant tissue containing the fungus, bypassing the need to culture and isolate the fungus separately. This extraction approach obtains fungal genetic material in situ from infected plant samples, overcoming the limitation of difficult in vitro cultivation and enabling molecular characterization without requiring pure fungal cultures.
Solution Approach 2:
The patent uses qPCR to create amplified copies of specific fungal DNA sequences from the original extracted DNA. This copying process generates sufficient quantities of target DNA sequences for analysis and detection, enabling genetic database construction and characterization without needing to maintain large amounts of live fungal cultures.
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 qPCR method allows for early and accurate detection of Erysiphe necator DNA on grapevine leaves, facilitating timely fungicide treatments and improving disease management by providing a reliable, molecular-based diagnostic tool.
Implementation Method 1
The method according to the invention makes use of the quantitative Polymerase Chain Reaction (PCR) technology, also known as qPCR or real-time PCR. PCR is the technology allowing the rapid amplification of target DNA sequences using specific oligonucleotides as primers of amplification reactions which take place in repeated cycles.
Implementation Method 2
Classical PCR allows the qualitative detection of certain target DNA sequences, whereas qPCR allows a quantitative measure of the amount of the target DNA sequence.
Data Source
AI summary
The present invention relates to means, methods and kits for the specific detection of the causing agent of powdery mildew on grapes, the fungus Erysiphe necator. More specifically, the methods according to the invention are quantitative methods based on quantitative Polymerase Chain Reaction.