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A strain-specific quantitative PCR detection kit and application of transgenic wheat b73‑6‑1

A genetically modified wheat, specific technology, applied in the direction of determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of lack of genetically modified wheat B73-6-1, low qualitative detection sensitivity, and inability to quantitative PCR detection. , to achieve the effect of good accuracy, good strain specificity and high sensitivity

Inactive Publication Date: 2018-01-16
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on the detection method of transgenic wheat B73-6-1 is limited to qualitative detection, but not quantitative PCR detection. At the same time, the sensitivity of qualitative detection is not high, and the quantitative PCR detection method of genetically modified wheat B73-6-1 is lacking in the prior art

Method used

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  • A strain-specific quantitative PCR detection kit and application of transgenic wheat b73‑6‑1
  • A strain-specific quantitative PCR detection kit and application of transgenic wheat b73‑6‑1
  • A strain-specific quantitative PCR detection kit and application of transgenic wheat b73‑6‑1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1 Analysis of the flanking sequence of transgenic wheat B73-6-1 foreign gene

[0037] 1.1 DNA extraction

[0038] The genetically modified wheat B73-6-1 and non-transgenic wheat are mixed according to the mass percentage to make eight different genetically modified content (10%, 5%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) sample. Weigh 100 mg of the sample material ground into powder, and extract the total DNA of the sample using a plant DNA extraction kit. The extracted total DNA is dissolved in 100μL ddH 2 In O, the concentration was measured by NanoDrop 2000 (Thermo Scientific, USA) ultraviolet spectrophotometer, and finally diluted to 100ng / μL for later use. The OD260 and OD280 of the DNA solution were measured with an ultraviolet spectrophotometer, and the ratio of OD260 / OD280 was calculated to evaluate the quality of the extraction. The OD ratios of the DNA used in this study were all around 1.9.

[0039] 1.2 DNA library construction

[0040] The kit used to construct the li...

Embodiment 2

[0084] Example 2: Detection of actual samples

[0085] 1. Sample preparation: select 6 wheat samples to be tested (Emai 18, Yumai 28, Jimai 22, Shannong 19, genetically modified wheat B73-6-1, genetically modified wheat B72-8-11b), and use plant DNA extraction The kit is used to extract wheat genomic DNA, and NanoDrop 2000 is used to detect the quality and concentration of the DNA to confirm that the sample DNA meets the experimental requirements.

[0086] 2. According to the method established in Example 1, 6 wheat samples were tested, and the results were as follows Picture 11 Shown.

[0087] From Picture 11 It can be seen from the test results that the established genetically modified wheat B73-6-1 strain-specific fluorescence quantitative PCR detection method can accurately, sensitively and specifically detect the amplification curve from the genetically modified wheat B73-6-1 sample, but not No amplification curve was detected in the transgenic wheat and transgenic wheat B7...

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Abstract

The invention discloses a transgene wheat B73-6-1 line specificity quantitative PCR detection kit and applications thereof, and belongs to the technical field of transgene crop detection. The provided kit comprises a specific primer and probe of B73-6-1 line and a primer and probe of an endogenous reference gene. The specific primer and probe of B73-6-1 line is designed according to the upstream flanking sequence of 5' end of B73-6-1. The endogenous reference gene is designed according to the GAG56D wheat alcohol soluble protein gene. The provided kit can carry out qualitative detection and quantitative detection, and has the advantages of good accuracy, high sensitivity, and excellent specificity, and the detection sensitivity can reach 0.01%.

Description

Technical field [0001] The invention relates to a strain-specific quantitative PCR detection kit for transgenic wheat B73-6-1 and an application thereof, and belongs to the technical field of transgenic crop detection. Background technique [0002] Transgenic wheat is wheat that can express the target gene after screening by introducing foreign genes into wheat genes using genetic engineering technology. Transgenic wheat B73-6-1 is a gene gun that combines pHMW-1Dx5 (carrying high-molecular-weight glutenin subunit HMW-GS gene, driven by its own endosperm-specific promoter) and pAHC25 (carrying marker genes bar and GUS genes, upstream connection wide Ubiquitin promoter) Two plasmids were co-transformed into common wheat varieties, and the homozygous transgenic materials expressing foreign genes HMW-GS, GUS and bar genes were obtained after screening. At present, the research on the detection method of genetically modified wheat B73-6-1 is limited to qualitative detection, not qua...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6851C12Q1/686
CPCC12Q1/6851C12Q1/6895C12Q2531/113C12Q2545/101
Inventor 张明辉于微高学军甄贞刘营仇有文
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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