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Primer set, kit and method for detecting maize mir162 by lamp

A detection method and primer set technology, applied in the field of molecular biology, can solve the problems of detection methods and kits for undiscovered transgenic maize MIR162 strains, achieve broad application value and market prospects, simple result determination, and short detection time Effect

Inactive Publication Date: 2016-01-13
陈双雅
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] There is no method and kit for applying LAMP to the detection of transgenic maize MIR162 lines

Method used

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  • Primer set, kit and method for detecting maize mir162 by lamp
  • Primer set, kit and method for detecting maize mir162 by lamp
  • Primer set, kit and method for detecting maize mir162 by lamp

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: the detection of corn sample

[0044] 1. Primer design and synthesis:

[0045] According to the gene sequence of the transgenic maize line MIR162 (Genbank sequence number HI203349), the LAMP primer set was designed. The LAMP primer set consisted of outer primer F3, inner primer FIP, inner primer BIP, outer primer B3, loop primer LF, and loop primer LB. The specific sequence is as follows:

[0046] Outer primer F3: 5'-ACATGTAATGCATGACGTTAT-3'SEQ ID No: 1

[0047] Outer primer B3: 5'-GCCTTATCTGTTGCCTTCA-3'SEQ ID No: 2

[0048] Inner primer FIP:

[0049] 5'-CGCTATATTTTGTTTTTCTATCGCGTGAGATGGGTTTTTTATGATTAGAGTC-3'

[0050] SEQ ID No: 3

[0051] Inner primer BIP:

[0052] 5'-AACTAGGATAAATTATCGCGCGCCGGACGTTTTTAATGTACTGAA-3'SEQ ID No: 4

[0053] Loop primer LB: 5'-TGTCATCTATGTTACTAGATCCCCG-3'SEQ ID No: 5

[0054] The above primers were synthesized by TAKARA Company. The outer primer F3 solution, the outer primer B3 solution of 10 μmol / L, the inner pri...

Embodiment 2

[0066] Example 2: Determination of the specificity of the LAMP method for the detection of transgenic maize MIR162 lines

[0067] Take 13 parts of seeds or fruits of other plant species, 14 parts of standard samples of different transgenic maize lines, and 1 part of non-transgenic maize. Genomic DNA was extracted according to the above method, and subjected to LAMP isothermal amplification, and the fluorescent dye SYBRGreenI colorimetric method was used to detect the results. The results showed that only the transgenic maize MIR162 line produced green specifically, and the test results were as follows: figure 1 and figure 2 shown.

[0068] See Example 1 for the primer sets and methods used.

[0069] figure 1 It is a diagram of the experimental results of detecting MIR162 and other 13 plant species by using the primer set and detection method of the present invention; Tubes 1-14 are sequentially transgenic corn MIR162 (10%, that is, the ratio of the mass of transgenic c...

Embodiment 3

[0072] Example 3: Determination of the sensitivity of the LAMP method for the detection of transgenic maize MIR162 lines

[0073] 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 100%, 10%, 5%, 1%, 0.5%, 0.1%, 0% Genomic DNA was extracted respectively according to the methods of the above-mentioned examples, and then subjected to isothermal amplification, and the results were detected by chromogenic method. Test results such as image 3 shown. image 3 It is a diagram of the sensitivity test results of the LAMP detection method of the transgenic maize MIR162 strain of the present invention. Tubes 1 to 7 are the LAMP test results of the corn seed samples with transgenic corn line MIR162 content respectively 100%, 10%, 5%, 1%, 0.5%, 0.1%, and 0%.

[0074] See Example 1 for the primer sets and methods used.

[0075] The experimental results showed that the samples wit...

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Abstract

The invention discloses a primer set, a kit and a method for detecting maize MIR162 by LAMP. The primers are composed of outer primer F3, outer primer B3, inner primer FIP, inner primer BIP and loop primer LB. The nucleotide sequence is shown in SEQ? ID? No: 1~5. The invention also protects the LAMP kit containing the primer. The detection method of the invention has the characteristics of quickness, simplicity, accuracy and sensitivity, has practical application value in the management of genetically modified products and inspection and quarantine work, and is especially suitable for the promotion and application of grass-roots laboratories and on-site detection.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a detection method of a transgenic plant strain, in particular to a primer set, a kit and a method for detecting maize MIR162 by LAMP. Background technique [0002] Since GM crops were commercially planted in 1996, economic and social benefits have gradually emerged. According to the research report of the International Service for the Application of Agricultural Biotechnology (ISAAA), the global planting area of ​​genetically modified crops reached 170 million hectares in 2012, an increase of 100 times compared with 1996 (1.7 million hectares). These figures reflect the global development trend of GMO breeding. However, because transgenic technology may produce new toxins and allergens, and cause gene drift of resistance genes, the international community still has considerable controversy over the safety of genetically modified foods. Many countries in the world, incl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q2531/119
Inventor 陈双雅王嘉鹤刘棠彭小莉许秋贝张永祥
Owner 陈双雅
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