Method for detecting transgenic event in plant

A technology of genetically modified events and events, applied in the fields of biotechnology and agricultural science, can solve the problems of cumbersome and complicated operation steps, high reagent and labor costs, etc.

Pending Publication Date: 2022-05-27
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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AI-Extracted Technical Summary

Problems solved by technology

This kind of analysis process generally uses technologies such as fluorescent PCR and digital PCR to perform parallel detection of multiple transgenic transforma...
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Method used

ABC three kinds of rapeseeds are mixed in non-transgenic seeds with lower ratio, and carry out shielding autofluorescence treatment. The test results showed that after shielding autofluorescence treatment, a lower...
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Abstract

The invention provides a method for improving the efficiency of detecting a transgenic event contained in a sample. The method comprises the following steps: labeling a first target antibody with a first fluorescent dye, and labeling a second target antibody with a second fluorescent dye; after sample treatment, paraffin embedding section, paraffin section dewaxing to water, antigen repair, autofluorescence shielding treatment, immunofluorescence staining and microscopic examination photographing, the number of transgenic events in a to-be-detected sample is judged according to the observed fluorescence color.

Application Domain

Biological material analysisBiological testing

Technology Topic

AntigenParaffin section +12

Image

  • Method for detecting transgenic event in plant
  • Method for detecting transgenic event in plant
  • Method for detecting transgenic event in plant

Examples

  • Experimental program(3)

Example Embodiment

[0036] Example 1 Detection of the number of transgenic events
[0037] Plant softening treatment
[0038] 1. Transfer the sample to a petri dish and dry it in a constant temperature oven at 37°C for 48h
[0039] 2. The sample is placed in the tissue grinder, and the tissue powder is collected after sufficient grinding
[0040] 3. Take 1g of powder sample, mix well, transfer it into EP tube and wrap the tissue powder with 3% agar, and put it into the agar after it cools and solidifies.
[0041] Fix in FAA fixative solution (50% concentration for young plants and 70% concentration for old hard tissues) for 24h.
[0042] 4. Take out running water and rinse for 2-4h
[0043] 5. For long-term storage, it can be transferred back to FAA fixative
[0044] Paraffin-embedded sections of samples 1. Materials: fresh tissues were fixed in FAA fixative for more than 24 hours. Take the tissue out of the fixative solution, trim the tissue at the target site with a scalpel in a fume hood, and place the trimmed tissue and the corresponding label in a dehydration box.
[0045] 2. Dehydration: Put the dehydration box into the hanging basket and dehydrate with gradient alcohol in sequence in the dehydrator. 75% alcohol 4h-85% alcohol 2h-90% alcohol 2h-95% alcohol 1h- absolute ethanol I 30min- absolute ethanol II 30min- alcohol benzene 5-10min- xylene I 5-10min- xylene II 5- 10min-wax I1h-wax II 1h-wax III 1h.
[0046]3. Embedding: The tissue soaked in wax is embedded in an embedding machine. First put the melted wax into the embedding frame, and before the wax solidifies, take the tissue out of the dehydration box and put it into the embedding frame according to the requirements of the embedding surface and attach the corresponding label. Cool at -20℃, after the wax solidifies, remove the wax block from the embedding frame and trim the wax block.
[0047] 4. Slicing: Place the trimmed wax block on a paraffin microtome to slice, with a slice thickness of 4-6 μm. Floating the slices Flatten the tissue on warm water at 40°C in a spreader, pick up the tissue with a glass slide, and place it in a 60°C oven to bake the slices. After the water is dried and the wax is baked, take it out and store it at room temperature for later use.
[0048] Dewaxed paraffin sections to water
[0049] Put the slices into xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, and distilled water for washing.
[0050] antigen retrieval
[0051] Tissue sections were placed in a retrieval box filled with EDTA antigen retrieval buffer (pH 9.0) for antigen retrieval in a microwave oven. After medium heat to boiling, switch off the power at an interval of 10 minutes from medium to low heat to boiling. During this process, the buffer should be prevented from over-evaporating, and the tablets should not be dried. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times with shaking on a destaining shaker, 5 min each time.
[0052] Immunofluorescence staining
[0053] Add fluorescently labeled antibodies (cy3-bar antibody and FITC-EPSPS antibody): Gently shake off the blocking solution, drop the primary antibody prepared in PBS in a certain proportion on the slices, and incubate the slices in a humid box at 4°C overnight. (Add a small amount of water to the wet box to prevent the antibody from evaporating);
[0054] DAPI counterstained nuclei: the slides were placed in PBS (pH 7.4) and washed three times with shaking on a destaining shaker, 5 min each time. After the sections were slightly dried, DAPI staining solution was added dropwise to the circle, and incubated at room temperature for 10 minutes in the dark;
[0055] Mounting: The slides were placed in PBS (pH 7.4) and washed three times with shaking on a destaining shaker, 5 min each time. After drying, the sections were mounted with anti-fluorescence quenching mounting medium.
[0056] Microscope photo
[0057] Sections were observed under a Nikon inverted fluorescence microscope and images were collected.
[0058] Result analysis
[0059] like figure 1.A green observed for PAT/bar protein, blue fluorescence of B cell nuclei (background fluorescence), C superposition of blue and green. If the sample observes as figure 1 As shown in C, it means that there is only PAT/bar protein in the sample, which means that the sample contains a transformation event.
[0060] figure 2.A Red observed for CP4-EPSPS protein, blue fluorescence of B cell nuclei (background fluorescence), C superposition of blue and red. If the sample observes as figure 2 As shown in C, it means that there is only CP4-EPSPS protein in the sample, which means that the sample contains a transformation event.
[0061] image 3.A green observed for PAT/bar protein, B red observed for CP4-EPSPS protein, C blue fluorescence of nuclei (background fluorescence), superposition of blue, red and green. If the sample observes as image 3 As shown in C, the sample contains two transformation events.

Example Embodiment

[0062] Example 2 Detection of Sensitivity
[0063] According to the experimental method in Example 1, prepare rapeseed A seeds containing only PAT/bar transgenic events, B rape seeds containing only CP4-EPSPS transgenic events, and C rapeseed seeds containing both CP4-EPSPS and PAT/bar transgenic events seed. The three kinds of rapeseed seeds ABC were mixed with non-transgenic rapeseed seeds at a ratio of 0.1%, and then tested.
[0064] The results showed that in the case of only 0.1% of transgenic seeds, the fluorescent immunoassay method could accurately identify the number of transgenes in the sample to be tested, and its detection sensitivity was 0.1%.

Example Embodiment

[0065] Example 3 Sensitivity detection after shielding autofluorescence treatment
[0066] The operation is performed according to the experimental method in Example 1-2. Specifically, after antigen retrieval, the operation of shielding autofluorescence is performed. The specific operation is as follows: adding autofluorescence quencher A and autofluorescence quencher B in sequence, and finally performing The serum is blocked; the autofluorescence quencher A is a mixture of pH 7.4 phosphate buffer and sodium borohydride; the autofluorescence quencher B is a mixture of Sudan black B and absolute ethanol; autofluorescence is added first Quencher A, incubate at room temperature for 10-20 min, and wash; then, add autofluorescence quencher B, incubate at room temperature for 10-15 min, wash, and then perform immunofluorescence staining and observation.
[0067] The three rapeseed seeds ABC were mixed at a lower ratio in non-transgenic seeds and subjected to a shielded autofluorescence treatment. The test results showed that a lower proportion of transgenic seeds could also be detected after the autofluorescence shielding treatment, and the sensitivity was improved; compared with the control without autofluorescence shielding, the detection sensitivity could be increased by about 10 times.

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