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Primer and method for detecting transgenic maize strain MIR162

A technique for transgenic corn and detection methods, applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve problems such as non-specific detection methods for MIR162 strains, and achieve good specificity and high sensitivity , quick and easy detection method

Inactive Publication Date: 2014-01-01
陈双雅
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the amplified products of common PCR primer pairs in the two patents are only 94bp and 98bp respectively, and too small target sequences are likely to lead to false positive results.
In addition, the Blast results of the primer sequences in the detection method of the patent application CN201210053592 in Genbank show that the target fragment is not the border sequence of the corn plant genome and foreign DNA fragments, so this method is not a specific detection method for MIR162 strains

Method used

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  • Primer and method for detecting transgenic maize strain MIR162
  • Primer and method for detecting transgenic maize strain MIR162
  • Primer and method for detecting transgenic maize strain MIR162

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: the detection of corn sample

[0039] 1. Primer design and synthesis:

[0040] According to the gene sequence of the transgenic maize line MIR162 (Genbank sequence number HI203349), primers were designed, consisting of forward primer 193F and reverse primer 751R, wherein the sequence of forward primer 193F is SEQ ID No: 1 and the sequence of reverse primer 751R is SEQ The specific sequence of ID No:2 is as follows:

[0041] SEQ ID No: 1: 5'-gagtcccgcaattatacat-3'

[0042] SEQ ID No: 2: 5'-ggtttgggcaaaatctcaaac-3'.

[0043] The above primers were purchased from TAKARA Company. Prepare solutions with a concentration of 10 μmol / L respectively as kits for future use.

[0044] Genomic DNA extraction:

[0045] For the DNA extraction method, please refer to "Molecular Cloning Experiment Guide, Third Edition" (written by Sambrook D.W Russell. Science Press 2002 [US] J. Huang Peitang et al. translation).

[0046] Amplification:

[0047] 3.1 PCR rea...

Embodiment 2

[0054] Example 2: Determination of the specificity of the PCR method for the detection of the transgenic maize line MIR162

[0055] Take 13 parts of seeds or fruits of other plant species, 14 parts of standard products of different transgenic maize lines, and 1 part of non-transgenic maize. Genomic DNA was extracted according to the above method, PCR amplification was performed, and the results were detected by gel electrophoresis. The results showed that only the transgenic maize line MIR162 specifically produced an amplified band of 559 bp, and the detection results were as follows: figure 2 and image 3 shown.

[0056] See Example 1 for primers and methods used.

[0057] figure 2 It is a diagram of the experimental results of detecting MIR162 and other 13 plant species using the primers and detection method of the present invention; lanes 1-14 are sequentially transgenic corn MIR162 (10%), non-transgenic soybeans, rice, potatoes, peas, mung beans, wheat, Carrot seed...

Embodiment 3

[0060] Example 3 : Sensitivity determination of a PCR method for detection of transgenic maize line MIR162

[0061] The transgenic corn line MIR162 standard was mixed with non-transgenic corn seeds to prepare the relative percentage (w / w) of the transgenic corn line MIR162 as 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01% Genomic DNA was extracted respectively according to the method of the above-mentioned embodiment, and then PCR amplification was carried out, and the results were detected by gel electrophoresis. Test results such as Figure 4 shown. Figure 4 It is a diagram of the sensitivity test results of the PCR detection method of the transgenic maize line MIR162 of the present invention. Swimming lanes 1-8 are the PCR detection results of the corn seed samples and non-transgenic corn seeds with the contents of 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, and 0.01% of the transgenic maize line MIR162 respectively.

[0062] See Example 1 for primers and methods used.

[0063] The exper...

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Abstract

The invention discloses a primer and a method for detecting a transgenic maize strain MIR162. The primer consists of a forward primer 193F and a reverse primer 751R, wherein the nucleotide sequence of the forward primer 193F is shown as SEQ ID No:1, and the nucleotide sequence of the reverse primer 751R is shown as SEQ ID No:2. The invention also discloses a kit comprising the primer. The detection method has the characteristics of high specificity, rapidness, simplicity, convenience, high accuracy and high sensitivity, has high actual application value in management and inspection and quarantine of transgenic products and is particularly suitable for popularization and application in local laboratories.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer and a method for detecting transgenic maize line MIR162. Background technique [0002] Genetically modified crops have developed rapidly in recent years, and more and more genetically modified plant products have been approved for commercial planting in various countries around the world. According to the latest research report of the International Service for the Application of Agricultural Biotechnology (ISAAA), in 2012, the global planting area of ​​genetically modified crops reached 170.3 million hectares, an increase of 6% over 2011 and a 100-fold increase over 1996 (1.7 million hectares). Among them, GM corn is the second largest GM crop in the world after GM soybean. Among all commercialized GM crops, GM maize lines had the most lines with a total of 65 transformation events. [0003] However, because transgenic technology may produce new toxins and allergens, and c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895
Inventor 陈双雅王嘉鹤许秋贝张永祥
Owner 陈双雅
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