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Double digital PCR fluorescent quantitative detection method for transgenic maize NK603

A technology for fluorescent quantitative detection and genetically modified corn, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve the problem that genetically modified detection is in its infancy, unsuitable for genetically modified component detection methods, and deviations in experimental results and other problems, to achieve the effect of simple and easy detection process, avoid adverse effects, and improve stability

Inactive Publication Date: 2015-12-02
CHINESE ACAD OF INSPECTION & QUARANTINE
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AI Technical Summary

Problems solved by technology

However, research on GMO detection using digital PCR is still in its infancy
The existing digital PCR detection methods all need to go through the sample pretreatment process. In the actual detection, the pretreatment process often leads to deviations in the experimental results.
Therefore, the existing digital PCR detection method is not suitable for direct detection of genetically modified components

Method used

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  • Double digital PCR fluorescent quantitative detection method for transgenic maize NK603
  • Double digital PCR fluorescent quantitative detection method for transgenic maize NK603
  • Double digital PCR fluorescent quantitative detection method for transgenic maize NK603

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1 Design of primers and probe combinations for double digital PCR fluorescence quantitative detection of transgenic maize NK603

[0033] According to the insertion sequence and border sequence of the exogenous gene of the transgenic maize NK603 strain, the following primer and probe combinations (SeqIDNo.1-6) for the dual digital PCR fluorescence quantitative detection of the transgenic maize NK603 were designed:

[0034] Foreign gene forward primer: 5'-ATGAATGACCTCGAGTAAGCTTGTTAA-3'

[0035] Foreign gene reverse primer: 5'-AAGAGATAACAGGATCCACTCAAACACT-3'

[0036] Foreign gene probe: 5'-FAM-TGGTACCACGCGACACACTTCCACTC-BHQ1-3'

[0037] Internal reference gene adh1-135 forward primer: 5'-CGTCGTTTCCCATCTCTTCCTCC-3'

[0038] Internal reference gene adh1-135 reverse primer: 5'-CCACTCCGAGACCCCTCAGTC-3'

[0039] Internal reference gene adh1-135 probe: 5'-VIC-AATCAGGGCTCATTTTCTCGCTCCTCA-BHQ1-3'.

Embodiment 2

[0040] Example 2 Establishment of double digital PCR quantitative detection method for transgenic maize NK603

[0041] 1.1 Experimental materials

[0042] The transgenic maize NK603 sample used in this example and its parental non-transgenic samples, transgenic maize lines BT176, MON810, MIR604, MIR162, 3272, and MON863 were all provided by the Chinese Academy of Inspection and Quarantine. The PCR chip was purchased from Fluidigm, USA, model number 48.770 DigitalArrayChip; the BioMarkHD high-throughput gene analysis system (BiomarkHD System) was purchased from Fluidigm.

[0043] 1.2 Genomic DNA extraction of transgenic crop seed samples for experiments

[0044] (1) The crop seed sample is ground and thoroughly air-dried;

[0045] (2) Mixing the transgenic corn line and parental non-transgenic seed samples in different proportions, and mixing overnight with a mixing instrument to obtain samples with different transgene concentrations for experiments;

[0046] (3) Weigh 100 m...

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Abstract

The invention provides a double digital PCR fluorescent quantitative detection method for transgenic maize NK603. According to the method, quantitative detection can be directly performed on the transgenic maize NK603 in genomes of samples to be detected, and therefore the standard curve plotting step in quantitative detection of a conventional PCR method and the sample preprocessing step in existing digital PCR detection are omitted; in addition, transgenic ingredients are calculated according to the proportion of the exogenous gene copy number and the reference gene copy number, and therefore the stability of quantitative detection of the transgenic ingredients can be improved by performing double detection on one reaction system. Through the method, absolute quantitative detection on the transgenic maize NK603 can be achieved, the quantitative detection limitation can reach 0.5%, the sensitivity can reach 0.1%, and the requirements of actual detection of all the transgenic ingredients can be met. In addition, the method is easy to operate and high in flexibility and serves as an effective method for absolute quantitative detection of transgenes.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection, in particular to a double digital PCR fluorescence quantitative detection method for transgenic corn NK603. Background technique [0002] Since the first transgenic tomato was successfully commercialized in the United States in 1996, the research on genetically modified crops has achieved rapid development in the past two decades. According to ISAAA statistics, as of 2014, the global planting area of ​​genetically modified crops has reached 180 million hectares, nearly 100 times higher than the planting area in 1996, accounting for 3.4% of the total global crop planting area. The rapid development of genetically modified crops not only provides more convenient breeding methods for modern agriculture, but also has attracted widespread attention from various countries and regions due to their unknown safety hazards. To this end, various countries and regions have established la...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6895C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2537/16C12Q2545/101
Inventor 付伟朱鹏宇杜智欣王晨光魏霜张亮亮彭萱子朱水芳
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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