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Specific primer and probe for assaying genetically modified corn MIR162 and application thereof

A technology of MIR162, transgenic corn, applied in DNA/RNA fragments, fluorescence/phosphorescence, recombinant DNA technology, etc., can solve the problems that have not been discovered, and achieve the effect of broad application value and market prospect

Inactive Publication Date: 2012-07-18
INST OF PLANT PROTECTION SHANDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After searching the existing patents and other documents, the applicant has not found any reports on the specific qualitative PCR of transgenic maize MIR162 transformants, SYBR Green I real-time fluorescent quantitative PCR and TaqMan probe real-time fluorescent quantitative PCR detection

Method used

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  • Specific primer and probe for assaying genetically modified corn MIR162 and application thereof
  • Specific primer and probe for assaying genetically modified corn MIR162 and application thereof
  • Specific primer and probe for assaying genetically modified corn MIR162 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Transgenic Maize MIR162 Transformant Specific Qualitative PCR Detection Method

[0045] Primers were synthesized by Dalian Takara Company and diluted to 10umol / L for use. The synthetic primer sequences are as follows:

[0046] MIR162-F: 5'-CTGTCTAATAGTTTGAGTGA-3';

[0047] MIR162-R: 5'-GTGACTCCCTTAATTCTC-3'.

[0048] Extract transgenic corn MIR162, MON810, NK603, Bt176, MON88017, GA21, TC1507, MIR604, Bt11, transgenic soybean GTS40-3-2, MON87701, non-transgenic soybean 1138-2, transgenic cotton MON88913, LLcotton25, MON531, MON1445, transgenic Rice TT51, Kefeng 6, and non-transgenic maize Zhengdan 958 gene DNA were used as templates, and PCR amplification was performed with MIR162-F and MIR162-R primer combinations, respectively. The PCR program was: 95°C 5min pre-denaturation; 94°C 30s, 50°C 30s, 72°C 30s, 35 cycles; 72°C extension 7min. PCR amplified products were separated by agarose gel electrophoresis, and the existence of amplified products was ident...

Embodiment 2

[0050] Example 2 Transgenic maize MIR162 transformant-specific SYBR Green I real-time fluorescent quantitative PCR detection method

[0051] Extract the genomic DNA of the transgenic maize MIR162 standard, and dilute it to 5 0 , 5 -1 , 5 -2 , 5 -3 , 5 -4 ; The corresponding copy number is set to 100000, 20000, 4000, 800, 160, and three repetitions are set for each concentration to detect the repeatability of the amplification.

[0052] Primers were synthesized by Dalian Takara Company and diluted to 10umol / L for use. The synthetic MIR162 primer sequence is as follows:

[0053] MIR162-F: 5'-CTGTCTAATAGTTTGAGTGA-3';

[0054] MIR162-R: 5'-GTGACTCCCTTAATTCTC-3'.

[0055] Maize internal standard primer (zSSIIb) adopts national standard primer, and its sequence is as follows:

[0056] zSSIIb-F: 5'-CGGTGGATGCTAAGGCTGATG-3';

[0057] zSSIIb-R: 5'-AAAGGGCCAGGTTCATTATTCTC-3'.

[0058] The amplification reaction volume is 20uL, 2×Premix Ex Taq (Rox) 10uL, Forward primer 0.4uL w...

Embodiment 3

[0063] Example 3 Transgenic maize MIR162 transformant-specific TaqMan probe real-time fluorescent quantitative PCR detection method

[0064] Extract the genomic DNA of the transgenic maize MIR162 standard, and dilute it to 5 0 , 5 -1 , 5 -2 , 5 -3 , 5 -4 ; The corresponding copy number is set to 100000, 20000, 4000, 800, 160, and three repetitions are set for each concentration to detect the repeatability of the amplification.

[0065] Primers and probes were synthesized by Dalian Takara Company and diluted to 10umol / L for use. The synthetic MIR162 primer and probe sequences are as follows:

[0066] MIR162-F: 5'-CTGTCTAATAGTTTGAGTGA-3';

[0067] MIR162-R: 5'-GTGACTCCCTTAATTCTC-3';

[0068] MIR162-P: FAM-5'-CAGATTGTCGTTTCCCGCCTTC-3'-Eclipse.

[0069] Maize internal standard primers and probes (zSSIIb) adopt national standard primers and probes, and their sequences are as follows:

[0070] zSSIIb-F: 5'-CGGTGGATGCTAAGGCTGATG-3';

[0071] zSSIIb-R: 5'-AAAGGGCCAGGTTCATTAT...

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Abstract

The invention discloses a specific primer for assaying genetically modified corn MIR162, the sequence of which is shown as SEQ ID NO:1 and SEQ ID NO:2. The invention also discloses a fluorescently labeled probe for assaying genetically modified corn MIR162, the sequence of which is shown as SEQ ID NO:3, the 5' end of the probe is connected with a fluorescent reporter FAM, and the 3' end of the probe is connected with a fluorescent quencher Eclipse. The invention also discloses an assay method which utilizes the primer and the probe to carry out gene-specific qualitative PCR (Polymerase Chain Reaction), SYBR Green I real-time fluorogenic quantitative PCR and TaqMan probe real-time fluorogenic quantitative PCR to assay whether corn and related products thereof contain MIR162 transformation events, and an experiment proves that the assay method is sensitive, accurate, simple and reliable, and has a high application value and a wide market prospect.

Description

technical field [0001] The invention relates to specific primers and probes for detecting transgenic corn MIR162, and its use in gene-specific qualitative PCR, SYBR Green I real-time fluorescent quantitative PCR and TaqMan probe real-time fluorescent quantitative PCR to detect whether corn and related products contain MIR162 Application in transformation events, belongs to the field of molecular biology. Background technique [0002] In the early 1990s, the first genetically modified food on the market appeared in the United States, which was a kind of preserved tomato. Over the next ten years, genetically modified products were born one after another, especially in food and medicine. Since the ecological safety and food safety of genetically modified products have been controversial, more than 40 countries and regions have successively established and implemented genetically modified labeling systems. With the establishment and continuous improvement of these systems, peo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 路兴波张广远孙红炜李凡杨淑珂
Owner INST OF PLANT PROTECTION SHANDONG ACAD OF AGRI SCI
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