Simultaneous ITS and Beta-Tubulin PCR Fungi Detection
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Solution Overview
Problem
Current methods for detecting fungi, such as morphology observation and PCR-based gene detection, face challenges including complexity, time-consuming cultivation, and low accuracy when identifying multiple types of fungi, especially when they are similar, leading to false positive reactions and requiring separate cultivation and DNA extraction for each type.
Innovation Solution
A method using both the ITS region and the β-tubulin gene for simultaneous amplification in a PCR reaction, with optimized primer concentration ratios, allows for accurate detection of various fungi types without separate cultivation or DNA extraction, utilizing a DNA chip for specific identification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If the ITS region alone is used for amplification, then a wide variety of fungi can be detected, but false positive reactions occur frequently lowering identification accuracy
Solution Approach 1:
The patent combines amplification of two different gene regions (ITS region and β-tubulin gene) into a single PCR reaction system. By merging these two amplification processes, the invention achieves both broad detection coverage and high identification accuracy, as the combined results from both regions provide cross-validation and reduce false positives while maintaining the ability to detect diverse fungal types.
2Measurement precision
If separate cultivation and DNA extraction is conducted for each fungi type, then accurate identification can be achieved, but the inspection process becomes complicated and time-consuming
Solution Approach 1:
The patent merges multiple fungi types into a single cultivation sample and performs simultaneous DNA extraction and amplification for multiple target regions in one reaction system. This consolidation eliminates the need for separate processing steps for each fungi type, significantly simplifying the inspection process while maintaining accurate identification through multi-region analysis.
Solution Approach 2:
The PCR reaction system is designed to universally amplify multiple different gene regions (ITS region and β-tubulin gene) simultaneously from mixed fungal samples. This universal amplification capability allows the system to handle diverse fungi types in a single reaction, eliminating the need for separate cultivation and DNA extraction procedures for each type.
3Measurement precision
If cultivation is conducted separately according to fungi type, then accurate detection can be achieved, but the inspection period is extended to about 14 days
Solution Approach 1:
The patent performs preliminary DNA extraction and amplification of multiple target regions simultaneously from mixed fungal samples, eliminating the need for separate cultivation steps. This preliminary processing approach significantly reduces the inspection period while maintaining detection accuracy through comprehensive multi-region analysis of the DNA samples.
4Measurement precision
If the β-tubulin gene alone is used for amplification, then specific type detection is possible, but false positive reactions may occur
Solution Approach 1:
The patent merges amplification of the β-tubulin gene with amplification of the ITS region in a single reaction system. This combination provides cross-validation between the two regions, where the β-tubulin gene provides specificity for type detection and the ITS region provides additional confirmation, thereby reducing false positive reactions while maintaining high specificity.
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
This approach enables high-accuracy detection of multiple fungi types with reduced false positives, simplifying the process by allowing simultaneous cultivation and DNA extraction, and enabling prompt and efficient inspection of fungal presence in mixed samples.
Implementation Method 1
a target region is amplified by a PCR (polymerase chain reaction) method
Implementation Method 2
fungi present in a sample is identified by means of a DNA chip, to which a probe which connects complimentarily to an amplified product is fixed
Data Source
AI summary
A method for detecting fungi includes amplifying DNA fragments containing target regions in fungal DNA to confirm the presence or absence of an amplified product. As the target regions, both of the ITS region and the β-tubulin gene are used, and by using a primer set for amplifying the β-tubulin gene and a primer set for amplifying the ITS region in a reaction solution for PCR for amplifying the target regions, both of the target regions are simultaneously amplified according to one or two or more types of fungi.