Transgene wheat B73-6-1 line specificity quantitative PCR detection kit and applications thereof

A transgenic wheat, specific technology, applied in the direction of microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of lack of transgenic wheat B73-6-1, low qualitative detection sensitivity, quantitative PCR detection and other problems , to achieve good accuracy, good strain specificity and high sensitivity

Inactive Publication Date: 2015-10-21
NORTHEAST AGRICULTURAL UNIVERSITY
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  • 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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  • Transgene wheat B73-6-1 line specificity quantitative PCR detection kit and applications thereof
  • Transgene wheat B73-6-1 line specificity quantitative PCR detection kit and applications thereof
  • Transgene wheat B73-6-1 line specificity quantitative PCR detection kit and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0037] 1.1 DNA extraction

[0038] The transgenic wheat B73-6-1 and non-transgenic wheat were mixed according to mass percentage to make eight different transgenic contents (10%, 5%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%) sample. Weigh 100 mg of the sample material ground into powder, and use the plant DNA extraction kit to extract the total DNA of the sample. The extracted total DNA was dissolved in 100 μL ddH 2 O, its concentration was measured by a NanoDrop 2000 (Thermo Scientific, USA) ultraviolet spectrophotometer, and finally diluted to 100 ng / 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 ratio of the DNA used in this study was all around 1.9.

[0039] 1.2 DNA library construction

[0040] The kit used for the constructio...

Embodiment 2

[0084] Embodiment 2: detect actual sample

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

[0086] 2, according to the method established in embodiment 1, 6 wheat samples are detected, and the results are as follows: Figure 11 shown.

[0087] From Figure 11 It can be seen from the detection results that the established transgenic wheat B73-6-1 line-specific fluorescent quantitative PCR detection method can accurately, sensitively and specifically detect the amplification curve from the transgenic wheat B73-6-1 sample, instead of No amplification curve was detected in transgenic wheat and transgenic wheat B72-8-11b samples, which was consist...

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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 its application, belonging to the technical field of transgenic crop detection. Background technique [0002] Transgenic wheat is the use of genetic engineering technology to introduce foreign genes into wheat genes, and the wheat that can express the target gene is obtained after screening. Transgenic wheat B73-6-1 was mixed with pHMW-1Dx5 (carrying high-molecular-weight glutenin subunit HMW-GS gene, driven by its own endosperm-specific promoter) and pAHC25 (carrying marker gene bar and GUS gene, upstream connection broad Spectrum ubiquitin promoter) two plasmids were transformed into common wheat varieties through co-transformation, and the homozygous transgenic materials expressing exogenous genes HMW-GS, GUS and bar genes were obtained after screening. At present, the research on the detection method of transgenic wheat B73-6-1 is limited to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
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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