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Nucleic acid quantitative detection kit for transgenic rice No.2 Ke-Ming-Dao (KMD) rice

A technology for transgenic rice and nucleic acid quantification, which can be used in the determination/inspection of microorganisms, DNA/RNA fragments, fluorescence/phosphorescence, etc., and can solve the problem of low detection value of transgenic components.

Active Publication Date: 2013-10-23
NAT INST OF METROLOGY CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, after some organisms insert foreign genes to form genetically modified organisms, some characteristics of the organisms will change, so the genome of the genetically modified organisms will be affected during extraction, and the concentration of the extracted foreign gene DNA is lower than its true concentration, resulting in genetically modified organisms. The detection value of the component (i.e. copy number of exogenous gene / copy number fraction of internal standard gene) is lower than its actual value

Method used

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  • Nucleic acid quantitative detection kit for transgenic rice No.2 Ke-Ming-Dao (KMD) rice
  • Nucleic acid quantitative detection kit for transgenic rice No.2 Ke-Ming-Dao (KMD) rice
  • Nucleic acid quantitative detection kit for transgenic rice No.2 Ke-Ming-Dao (KMD) rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1 Screening of internal standard gene for detection of transgenic rice Kechidao No. 2 and optimization of fluorescent quantitative PCR reaction system

[0087] 1. Materials and methods

[0088] 1. 100% transgenic rice Kemodao No. 2 rice seed powder (from China Institute of Metrology)

[0089] 2. Synthesize the primers of 5 rice internal standard genes including GOS9, PLD, SPS, RBE4 and UBQ5 (the sequence information comes from the article "Molecular analysis and quantitative detection of a Transgenic rice line expressing a bifunctional fusion TPSP" and the primers of the exogenous gene Kechadao 2 (provided by China Jiliang Institute) are shown in Table 4.

[0090] 3. Using the extracted DNA No. 2 as a template, use 5 internal standard gene primers of rice and primers of the exogenous gene No. 2 to carry out PCR reaction respectively. The set system is: TaqMan Universal PCR Master Mix 12.5ul, 1ul of upstream primer (400nM), 1ul of downstream primer (400nM), 5ul...

Embodiment 2

[0117] Example 2 Quantitative Detection of Components of Transgenic Kechidao No. 2 in Rice Powder

[0118] 1. Materials and methods

[0119] 1. The sample to be tested is the standard substance of rice seed powder of transgenic rice Kechidao No. 2 with a mass fraction of 2% (developed by China Institute of Metrology)

[0120] 2. Accurately weigh the sample to be tested and the quality control product (the standard substance of the transgenic rice Kemodao No. 2 rice seed powder with a mass fraction of 1% and 5% content) with a balance

[0121] 3. Using the Plant Genome Extraction Kit Genomic DNA Purification Kit (Promega, USA) was used to extract the genomic DNA of the sample to be tested and the quality control product, and the purity and concentration of the extracted DNA were determined with a UV spectrophotometer and a Picogreen kit, respectively.

[0122] 4. The standard (containing about 10 6 、10 5 、10 4 、10 3 、10 2 Copy / ml Plasmid Molecular Standard Substances of...

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Abstract

The invention relates to a nucleic acid quantitative detection method and a detection kit for transgenic rice No.2 Ke-Ming-Dao (KMD) rice. According to the method and the kit disclosed by the invention, an endogenous reference gene RBE4 which has an optimal effect and is suitable for detection is screened out, and a PCR (polymerase chain reaction) system suitable for quantitative detection is found, so that the precise content of a transgenic component in the transgenic rice No.2 KMD rice or a product thereof can be rapidly and correctly obtained. The invention also relates to a method for correcting the bias between a detection value and a real value in transgenic organism quantitative detection. According to the method, the ratio of nucleic acid extraction efficiencies of a transgenic organism and a receptor non-transgenic organism is calculated to be used as a correction coefficient, thus the real correct content of the transgenic component can be obtained.

Description

Technical field: [0001] The invention relates to a nucleic acid quantitative detection method and a detection kit of the transgenic rice Kechidao No. 2. The invention also relates to a method for correcting the deviation between the transgene detection value and the real value, specifically, a method for establishing a correction coefficient for calculating the deviation correction. Background technique: [0002] The transgenic rice Kechidao 2 is an insect-resistant rice bred by transferring the exogenous gene Cry1Ab from the recipient rice Xiushui 11. [0003] There are the following reports on the transgenic detection method of Kemodao 2: Yue Yunfeng et al. published the article "Research on the Qualitative PCR Detection Method of Marker Genes in Transgenic Plants" published in the "Chinese Journal of Oil Crops", which reported the qualitative detection method of Kemodao. But so far, there is no accurate quantitative detection method for the transgenic rice Kechidao 2. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11G01N21/64
CPCC12Q1/6806C12Q1/6851C12Q2537/165C12Q2531/113C12Q2545/101
Inventor 王晶隋志伟李亮余笑波臧超董莲华
Owner NAT INST OF METROLOGY CHINA
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