Method for detecting transgenic plant and products thereof

A technology of transgenic plants and detection methods, applied in the field of rapid detection of transgenic plants and their products, can solve the problems of difficulty in determining which antibody detection to choose, taking a long time, inability to detect dry and dead materials, etc., and achieve detection results. Accurate effect

Inactive Publication Date: 2002-12-18
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

But in the past, people mainly aimed at the target gene, reporter or marker gene. The limitation is that if a specific plant does not know which gene the researcher introduces, and if a product contains multiple plant components, the above method will have great risks. Blindness, it takes a long time to determine whether it (they) are genetically modified plants or products
[0008] (2) RNA molecular hybridization (Northern hybridization), reverse transcription PCR (RT-PCR) and other analysis techniques are used to detect the expression status of the target gene at the RNA level. The defects are the same as above, and the test samples must be fresh. Plant materials, unable to detect ...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: PCR detection of transgenic papaya using T-DNA left and right border sequences and nos terminator sequence primers

[0028] As mentioned above, T-DNA left and right border sequences and nos termination sequences exist in most transgenic plants, so using these two sequences to synthesize primers for PCR is suitable for the detection of most transgenic plants and their products. In our past work, we obtained a virus-resistant papaya line transfected with the replicase (RP) gene of papaya ringspot virus (PRV), and carried out transgenic detection on it by PCR method.

[0029] (1) DNA was extracted from leaves of transgenic papaya by CTAB method.

[0030] (2) Synthesize a pair of primers, one of which is a T-DNA left and right border sequence primer (4-23nt): 5'-caggatatattggcgggtaa-3', and the other is a nos termination sequence primer (nos termination sequence 81-100nt): 5'-catgcttaacgtaattcaac-3'.

[0031] (3) Carry out PCR amplification reaction on sample D...

Embodiment 2

[0033] Example 2: Real-time quantitative PCR using T-DNA left and right border sequences and CaMV35S promoter primers

[0034] Detecting genetically modified tobacco

[0035] The CaMV 35S promoter has a strong plant promoter function and has no organ specificity, so it is widely used. So far, more than 70% of transgenic plants use this promoter. However, a small number of cruciferous plants are susceptible to CaMV infection. Therefore, when detecting such plants and their products, if the CaMV 35S promoter primers are used alone, important errors may be caused. Using the left and right border sequences of T-DNA and the CaMV 35S promoter respectively PCR with two primers can overcome this problem. The transgenic glyphosate-resistant tobacco obtained by us was detected by real-time quantitative PCR.

[0036] (1) Extract DNA from transgenic tobacco leaves by CTAB method.

[0037] (2) Synthesize a pair of primers, one of which is a T-DNA left and right border sequence ...

Embodiment 3

[0040] Example 3: PCR-ELISA detection of transgenic tomato using CaMV35S promoter and nos terminator sequence primers

[0041] We combined the Saliva-binding region (SBR) gene of Streptococcus mutans surface protein PAC (Streptococcus mutans surface protein) and its chimeric gene with Vibrio cholerae B subunit (Cholera toxin B subunit, CTB), Transformed tomato was co-cultivated to obtain transgenic plants, which were detected by PCR-ELISA method.

[0042] (1) Extract DNA from transgenic tomato fruit by conventional CTAB method.

[0043] (2) Synthesize a pair of primers, one of which is the CaMV35S promoter primer (the 61-70nt, 5' end connected to biotin): 5'-biotin-cagcaggtctcatcaagacg-3', and the other is the nos terminator sequence primer ( nos termination sequence 81-100nt complementary sequence): 5'-gttgaattacgttaagcatg-3'.

[0044] (3) Carry out PCR amplification reaction on sample DNA according to conventional methods, use pROSB plasmid DNA (including CaMV35S promoter,...

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PUM

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Abstract

The present invention relates to a transgenic plant and detection method of its product. Said invention uses the characteristic sequence being in transgenic plant: T-DNA left anjd right boundary sequence, CaMV 35S promotor, CaMV 35S poly A, nos promotor and nos termination sequence to design and synthesize initiator to make PCR to obtain correspondent DNA amplification product or signal of amplification product. According to the PCR result it can be used for recognizing that said detected sample is transgenic plant and its product or not.

Description

technical field [0001] The invention belongs to the technical field of transgenic plant safety management and the field of biotechnology, and in particular relates to a rapid detection method for transgenic plants and products thereof. Background technique [0002] 1. Safety management of transgenic plants [0003] A transgenic plant refers to a plant variant that artificially transfers an exogenous gene into the plant genome through a certain method, and then inherits and expresses it. The exogenous gene transferred into the plant genome is called a transgene. [0004] In 1983, humans first reported the birth of genetically modified tobacco. By 2000, the global planting area of ​​genetically modified plants had reached 44.2 million hectares. Since the birth of genetically modified plants, some people have worried about their possible ecological and food safety problems, and these concerns have been supported by some research results. In order to st...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 叶长明蓝崇钰魏祥东陈东红林周
Owner SUN YAT SEN UNIV
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