Transgenic product nucleic acid level detection process
Introduction to the testing process of genetically modified products
The issue of genetically modified foods has always been a controversial topic; how to scientifically and correctly detect genetically modified products has become a matter of great concern to government testing agencies and related workers.
The detection of genetically modified agricultural products is to detect whether the agricultural product contains foreign genes, that is, the promoter, the terminator, the marker gene, the target gene, and the abundance of the foreign gene transcription product. In terms of methods, it is further divided into detection of gene levels and detection of protein levels.
The process of nucleic acid level detection of genetically modified products is as follows:
The first step: sample preparation
A certain mass of the sample is weighed, ground in a mortar or treated with an automatic tissue homogenizer to obtain a sample powder, which simultaneously breaks the tissue cells and releases the nucleic acid.
Step 2: DNA extraction and purification
DNA templates can be prepared manually or by automated nucleic acid extraction purification systems using commercial DNA extraction kits for subsequent PCR analysis.
The third step: detection of endogenous genes
PCR detection of endogenous genes of species is mainly used to verify the quality of DNA template extraction;
The target endogenous gene can be amplified by ordinary PCR method, separated by agarose gel electrophoresis, and the gel imaging apparatus is used to observe whether there is an amplified band of the expected size;
The presence or absence of a target endogenous gene can also be directly detected by real-time fluorescent quantitative PCR.
A positive test result indicates that DNA suitable for detection can be extracted from the sample, and foreign gene detection can be performed. Otherwise, DNA extraction and purification should be performed again.
The fourth step: screening genetic testing and strain identification testing
For the detection of transgenic components in plants, first screen the CaMV 35S, NOS, NPTII, PAT, BAR genes (promoter, terminator, etc.) to determine whether they contain foreign genes; if the screening results are negative, report the results directly;
If the screening result is positive, it is necessary to further identify structurally specific genes or strain-specific genes for detecting MON810, Bt11, Bt176, T14/T25, CBH351, GA21, TC1507, MON863, NK603, Bt10 (structural genes) to determine what Transgenic lines.
Both of these steps can be performed by ordinary PCR or real-time fluorescent quantitative PCR.
Step 5: Confirm the experiment
If the common PCR method is used to detect the result of screening gene and structure-specific gene or strain-specific gene, it is necessary to confirm the result by real-time fluorescent PCR.
Step 6: Results Judgment and Calculation
The Ct value of the exogenous gene in the sample to be tested is ≥40, and the Ct value of the endogenous gene is ≤24. If the control is normal, it can be determined that the XXX gene is not detected in the sample; The Ct value of the exogenous gene in the sample to be tested is ≤36, and the Ct value of the endogenous gene is ≤24. If the control result is normal, the XXX gene can be determined by the sample; The Ct value of the exogenous gene in the sample to be tested is between 36 and 40, and real-time fluorescent PCR amplification should be repeated. Quantitative calculation: Using the positive standard and the gene detection Ct value, the copy number of the foreign gene and the endogenous gene sequence can be calculated separately, and the transgenic components can be quantitatively calculated according to the following formula:  |
Step 7: Expression of results
Qualitative results are expressed:
For X species, no transgenic components were detected;
For the X species, the transgenic component was detected and (further reported) the sample contained the XX transgenic line.
Quantitative results are expressed:
No transgenic components were detected, and the detection limit of the quantitative method was X%;
The XX transgenic component in the sample is lower than the detection limit of the quantitative method (X%);
The XX transgenic component contained in the sample is X%.
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