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Gene chip detection method of transgenic maize

A technology of transgenic corn and gene chips, applied in the field of detection, can solve problems such as false negatives, reduced detection sensitivity, and the inability to detect multiple samples at the same time

Inactive Publication Date: 2011-07-06
威海华康新材料科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But it also has its limitations: (1) The detection range is narrow, and the genetically modified food needs to contain the range to be detected, such as EPSPS gene herbicide-tolerant soybeans, Bt gene-transformed insect-resistant corn, etc., so it can only be tested in commercial GMF varieties. use in the case of
At present, commercial ELISA kits can only detect a few kinds of GMF, and a kit is only aimed at a specific transgenic product, so it cannot detect food samples with multiple mixed components with high throughput and fast; (2) it is prone to false positives. Negative results. On the one hand, the content of "new protein" in genetically modified products is usually very low, which is difficult to detect. On the other hand, proteins are volatile during food processing. Proteins in processed foods are likely to lose the antigenic epitopes targeted by antibodies. resulting in false negative ELISA test results
In addition, new modifications of proteins before and after expression in the recipient organism's genome can also lead to reduced detection sensitivity and false negative results
[0013] As far as the ELISA and PCR technologies commonly used in the detection of genetically modified products are concerned, the biggest disadvantage is the narrow detection range, low efficiency, and the inability to simultaneously detect multiple samples with high throughput and large scale, especially in the case of ignorance of the genetically modified background. Under these conditions, it is almost impossible to screen each candidate gene sequence or protein one by one.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033]For the sequences of 7 exogenous genes commonly used in transgenic maize and control genes (18SrRNA, Invertase endogenous gene), primers and probes were designed by primer design software primer5.0 and oligo6.0, and nucleotide Sequence alignment, from which primers and probes that meet the requirements are selected, and the information of the selected primers and probes is shown in Table 1 and Table 2.

[0034] Table 1

[0035] Primer number

Primer sequence

target DNA

1

TAA TAG AGC AAT GAA CAG TCG G

18SrRNA

2

CTT TCA ACA ACG GAT CTC TTG G

3

GGC CGG ATC GTC ATG CTC TAC A

Invertase endogenous gene

4

TTG GCG TCC GAC TTG ACC CAC T

5

GAT TCC ATT GCC CAG CTA TCT G

CaMV35S promoter

6

TAG AGG AAG GGT CTT GCG AAG G

7

GAA TCC TGT TGC CGG TCT TG

T-nos terminator

8

CGA TT CCC GAT CTA GTAAC

9

CGT CTT GAA GGT CGT GGTA

Modified ...

Embodiment 2

[0044] Using a fully automatic chip spotter, it is designed into a 6×9 array, including 18 detection target probes, two negative controls, 4 points for each pair of probes, and a gene for each row. The setting on the chip in Anhui A human gene that has no homology with the detection probe was used as a negative control to exclude the interference of non-specific hybridization during the hybridization process.

Embodiment 3

[0046] The established transgenic corn gene detection chip was used to repeatedly test and verify 20 standard samples. The results showed that only the corresponding internal reference gene (18SrRNA, invertase endogenous gene) hybridization detection signal appeared in the standard sample without the transformed gene, 0.1% The maize not only obtained the hybridization detection signal of the internal reference gene, but also obtained the hybridization detection signal of the specific exogenous gene. The verification results showed that the designed chip had very high sensitivity, and the accuracy of the experimental results was very high. The selected primers, probes Needle and reaction conditions are optimal.

[0047] The established transgenic corn gene detection chip was used to detect 10 unknown corn samples. The results showed that 18SrRNA, invertase, insect-resistant zSSIIb gene, insecticidal protein CryIAb gene, CaMV 35S promoter, T- The nos terminator and herbicide-res...

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Abstract

The invention discloses a gene chip detection method of transgenic maize. The main steps comprise primer and probe determination, chip preparation, PCR (polymerase chain reaction) amplification, PCR product marking, chip hybridization, chip scanning and data processing. By adopting the detection method, sequences of seven main exogenous DNAs (deoxyribonucleic acid) commonly used in transgenic maize, such as promoters, terminators, insect-resistant genes, glyphosate-resistant genes and the like, can be detected and analyzed through one step, so that the detection method of transgenic maize has the advantages of high throughput, miniaturization, automation, rapidity, high efficiency and high sensitivity, and can be widely applied to related fields such as import-export inspection and quarantine, and the like.

Description

technical field [0001] The invention relates to a detection method, specifically a method for detecting transgenic corn products by using a gene chip. Background technique [0002] With the rapid development of genetic engineering technology, genetically modified crops have entered the stage of large-scale commercial production. There are more than 100 varieties of genetically modified crops, mainly including soybeans, corn, cotton, rape, etc., and genetically modified foods and food ingredients processed and produced from genetically modified crops There are more than 4000 species. However, while genetically modified crops bring huge economic benefits, there are also many potential problems. Since there is still no scientific basis to prove the safety of genetically modified products to human health and the environment, the potential risks of genetically modified products to health and the environment have aroused great concern and widespread concern from governments and t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 孙炜伟杜蕾蕾
Owner 威海华康新材料科技有限公司
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