Digital PCR Primer-Probe Composition for CRM-Free GM Quantification
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Solution Overview
Problem
Existing PCR methods for relative quantification of GM crops require certified reference material (CRM), which is not always available, and suffer from amplification efficiency issues due to nucleic acid quality differences.
Innovation Solution
Use a gene present in single copy or 2 to 3 copies within the GM crop genome as a replacement for CRM, employing a primer and probe set for digital PCR to achieve relative quantification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If certified reference material (CRM) is used for relative quantification of GM crops, then quantification accuracy can be achieved, but the method becomes inapplicable when CRM is not available and suffers from amplification efficiency variations
Solution Approach 1:
The patent creates a virtual reference system by copying the endogenous gene sequence information from public databases (like Phytozome) to generate in silico CRM. This virtual CRM serves the same function as physical CRM but can be generated for any GM crop without requiring actual reference materials, thus resolving the contradiction between measurement precision and adaptability.
Solution Approach 2:
The patent introduces an endogenous gene as an intermediary reference that mediates between the introduced GM gene and the quantification process. This endogenous gene serves as a stable internal control that eliminates the need for external CRM while maintaining quantification accuracy, and it can be applied universally across different GM crop types.
2Measurement precision
If physical CRM is used for standard curve generation, then relative quantification can be performed, but nucleic acid quality differences cause amplification efficiency variations
Solution Approach 1:
Instead of using physical CRM that may have degraded or variable nucleic acid quality, the patent copies the reference gene sequences in silico from reliable public databases. This virtual copying eliminates nucleic acid quality issues entirely while maintaining the ability to generate standard curves for accurate relative quantification.
Solution Approach 2:
The patent replaces the physical mechanical system of handling and storing CRM materials with a digital information system. By substituting physical nucleic acid references with digital sequence data, the method eliminates problems related to nucleic acid degradation, storage conditions, and handling variability.
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 accurate relative quantification of GM crops without CRM, reducing measurement errors and amplification inefficiencies, suitable for GM crop quarantine and food inspection.
Implementation Method 1
digital PCR is a third-generation PCR (polymerase chain reaction) method
Implementation Method 2
carrying out emulsion PCR
Implementation Method 3
a fluorescent probe into one well and carrying out emulsion PCR. Subsequently, wells showing a fluorescent signal indicate that a sample with gene copy number of 1 has been applied and amplified
Data Source
Figure 1~2
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AI summary
The present invention relates to a method for relative quantification of GM (genetically modified) crops using digital PCR without the need for certified reference material (CRM). More specifically, the present invention relates to a primer and probe set composition for digital PCR to have relative quantification of an introduced gene in GM crops without the need for CRM, comprising a first primer and probe set for specific detection of a gene present in single copy or copy number of 2 to 3 in genome of the GM crops; and a second primer and probe set for specific detection of an introduced gene in the GM crops, and also a use of the primer and probe set composition.