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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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI 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 transformation events in a single seed in a large number of mixed samples, so it requires cumbersome and complicated steps and high reagent and labor costs. ideal analysis

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[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 hoo...

Embodiment 2

[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%.

Embodiment 3

[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 autofluores...

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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.

Description

technical field [0001] The invention relates to a simple and intuitive detection method for transgenic crops, in particular to a detection method for transgenic crops with complex traits, and belongs to the fields of biotechnology and agricultural sciences. Background technique [0002] Since the large-scale cultivation of genetically modified crops in 1996, more and more transformation events have been applied to different crops to meet people's needs for food, feed, clothing and other daily necessities. However, these transformation events must be officially approved in accordance with relevant local laws and regulations before they can be circulated in a country. In recent years, with the continuous emergence of new transformation events, there has been a trend of gene stacking of new transformation events with existing excellent transformation events. As of 2014, transgenes with complex traits caused by gene stacking have been Crop planting area has accounted for 28.3% ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/58
CPCG01N33/582G01N33/6854G01N2333/415
Inventor 高鸿飞吴刚文璐珂翟杉杉崔丹丹武玉花闫晓红李俊
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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