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Specific identification for transgenic rice kefeng No.6 strain via adoption of RPA (Recombinase Ploymerase Amplification) technology

A technical identification and transgenic technology, which is applied in the field of primer and probe combination, can solve the problems that cannot be further satisfied with the rapid detection of transgenic products, and achieve the effect of high sensitivity and improved ability

Active Publication Date: 2014-01-22
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, among the reported detection methods of genetically modified plants, the PCR instrument is mainly used for routine detection in the laboratory, and this method cannot further meet the rapid detection of genetically modified products.
At present, RPA technology has not been used for strain-specific identification of transgenic rice Kefeng 6 at home and abroad.

Method used

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  • Specific identification for transgenic rice kefeng No.6 strain via adoption of RPA (Recombinase Ploymerase Amplification) technology
  • Specific identification for transgenic rice kefeng No.6 strain via adoption of RPA (Recombinase Ploymerase Amplification) technology
  • Specific identification for transgenic rice kefeng No.6 strain via adoption of RPA (Recombinase Ploymerase Amplification) technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Design and screening of primers

[0018] First of all, primer design and screening: RPA primer design usually needs to consider the following factors: (1) GC content is 40%-60%; (2) Try to avoid secondary structures inside the primer; (3) Avoid primer duplication sequence. RPA requires a primer length of 30-35nt. In the experiment, multiple pairs of primers need to be designed from both ends of the target sequence for optimization, screening, and the replacement or increase or decrease of individual bases will have an important impact on the experimental results. In this experiment, a probe was designed according to the specific sequence of Kefeng 6 transformant (GenBank No. HM124448), and 4 upstream primers and 5 downstream primers were designed on both sides of the probe. Using 500 copies of Kefeng 6 genomic DNA as a template, different primer combinations were screened for fluorescence, and finally a pair of primers with short amplification take-off tim...

Embodiment 2

[0022] Example 2: Detection of Kefeng 6 with designed primers

[0023] 1. Experimental Materials

[0024] (1) Plant material

[0025] Genetically modified rice Kefeng No. 6 (leaf), genetically modified rice Kefeng No. 6 seed powder (content 5%), genetically modified rice Kefeng No. 6 seed powder (content 1%), genetically modified rice Kefeng 1 (leaf), genetically modified rice TT51-1 (leaf), transgenic rice Kangyou 97 (leaf), non-transgenic rice Minghui 63 (leaf).

[0026] (2) Enzymes and reagents

[0027] Molecular biology reagents, TwistAmp DNA Amplification Exo Kits were purchased from TwistDX Company, and other biochemical reagents were imported or domestic analytically pure. Primers were synthesized by Beijing Sangon Biotechnology Co., Ltd.

[0028] (3) Experimental equipment

[0029] DNA processing instrument: low temperature mixing ball mill MM400 (Retsch)

[0030] Fluorescence detector: RPA amplification detector (Twista) or fluorescent quantitative PCR instru...

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Abstract

The invention firstly provides an RPA (Recombinase Ploymerase Amplification) detection method of identification of transgenic insect-resistant rice kefeng No.6 strain and in particular discloses a primer and probe combination used for indentifying transgenic insect-resistant rice kefeng No.6 via a recombinase ploymerase amplification technology; a sequence of a positive primer is expressed as SEQ ID No.1; a sequence of a negative primer is expressed as SEQ ID No. 2; a sequence of the probe is expressed as SEQ ID No.3. The invention also discloses a method for indentifying transgenic insect-resistant rice kefeng No.6. The method comprises the following steps: extracting DNA (Deoxyribose Nucleic Acid) of a sample to be detected as a template, performing RPA quick amplification and real-time fluorescence detection by using the primer; and proving that the detected sample contains components of the transgenic insect-resistant rice kefeng No.6 if an obvious amplification curve is obtained.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a method for identifying the specificity of transgenic insect-resistant rice using a recombinase polymerase isothermal amplification technology (Recombinase Polymerase Amplification, RPA), and a corresponding primer and probe combination. Background technique [0002] At present, DNA amplification is the main method of nucleic acid detection. The commonly used PCR detection requires sophisticated instruments and cumbersome test procedures, which is difficult to meet the requirements of on-site detection in non-laboratory environments. In recent years, nucleic acid constant temperature amplification technology has been greatly developed. Compared with traditional PCR, nucleic acid constant temperature amplification technology does not require expensive PCR equipment, and can quickly amplify the target fragment in a short time. It is simple, fast, sensitive, etc. advantage. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q2521/101
Inventor 金芜军宛煜嵩徐潮李亮苗朝华黄卫红
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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