A kit and detection method for accurate and quantitative detection of transgenic maize line t25

A technology for quantitative detection of genetically modified corn, applied in the field of molecular biology, can solve the problems of unknown effectiveness and practicability, and a small number of research reports, and achieve the effects of reduced detection cost, high tolerance, and easy standardization

Active Publication Date: 2019-06-14
BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the quantitative detection of transgenes by ddPCR is still in its infancy, and the number of research reports is not much, mainly for Mon810 transgenic maize.

Method used

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  • A kit and detection method for accurate and quantitative detection of transgenic maize line t25
  • A kit and detection method for accurate and quantitative detection of transgenic maize line t25
  • A kit and detection method for accurate and quantitative detection of transgenic maize line t25

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 ddPCR primer, probe design

[0033] In order to realize the specific detection and absolute quantitative analysis of the transgenic maize line T25 and the maize genome, we selected the junction region between the 3' end of the inserted pat gene and the CaMV35S terminator (Genebank no.DQ156557.1) and the single-copy endogenous maize gene The 3' end of zein (GenBank no.JQ083080.1) was used as the target sequence, and sequence analysis and alignment were performed through NCBI online tools, and more than 50 pairs were designed using Prime Express software V3.0 (ABI, Foster City, CA, USA) Primer and probe combinations were screened to obtain one set of internal and external primers / probe combinations with strong specificity and suitable for ddPCR amplification conditions. The sequences are shown in Table 1. The 5' end of the exogenous gene pat-T35S probe is labeled with HEX, the 5' end of the endogenous gene zein probe is labeled with FAM, and the 3' ends of th...

Embodiment 2

[0034] Embodiment 2 kit composition

[0035] The kit includes primers and probes designed in Example 1 for detecting the transgenic maize line T25, a positive control (T25 transgenic maize leaf genomic DNA, a certified reference material, purchased from the American Society of Oil Chemists), ddPCRmaster mix (purchased from BioRad, USA), droplet generating oil (purchased from BioRad, USA), 25mM magnesium acetate (purchased from Sigma, USA), microdroplet generating card (purchased from BioRad, USA), Twin Tec Semi-Skirted 96-well plate (purchased from Eppendorf, Germany), and aluminum foil heat-sealing film (purchased from BioRad, USA).

Embodiment 3

[0036] Example 3 Establishment of detection method

[0037] 1. DNA extraction

[0038]The DNA in the samples was extracted by the modified CTAB method. The specific steps are as follows: take 100 mg sample, add 300 μL sterilized deionized water, and mix thoroughly; add 700 μL CTAB buffer solution preheated to 65 °C, add 10 μL RNase solution after mixing, shake; heat at 65 °C for 30 min; Add 10 μL proteinase K solution, mix gently, heat at 65°C for 30 minutes; centrifuge at 12000g for 10min, transfer the supernatant to a 1.5mL centrifuge tube containing 500μL chloroform, shake for 30s; centrifuge at 12000g for 15min until obvious stratification occurs; Transfer the upper liquid phase to a 1.5mL centrifuge tube containing 500μL chloroform, shake; centrifuge at 12000g for 5min, transfer the upper liquid phase to a new 1.5mL centrifuge tube; add 2 times the volume of CTAB precipitation buffer, pipette to mix Uniform; place at room temperature for 60min; centrifuge at 12000g for ...

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PUM

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Abstract

The invention discloses a kit for accurately and quantitatively detecting a transgenic maize line T25 and a detection method thereof. The invention in particular relates to a group of primers and probes which are used for detecting the transgenic maize line T25 as well as a kit containing the primers and probes, wherein the primers and probes have nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.6 in a sequence table. The invention also provides a detection method for accurately and quantitatively detecting the transgenic maize line T25 by utilizing a dual microdroplet type digital PCR technology. The detection method provided by the invention is independent of a certified reference material or other standard substances, has higher accuracy, sensitivity and repeatability and is easy to be standardized.

Description

technical field [0001] The invention relates to a kit and a detection method for quantitatively detecting transgenic corn strain T25, belonging to the field of molecular biology. Background technique [0002] With the widespread planting of genetically modified crops around the world, their food safety and environmental safety issues have been highly valued by consumers, governments and relevant international organizations. Many countries and regions have formulated safety management regulations on genetically modified organisms and implemented labeling systems. On January 5, 2002, the Ministry of Agriculture of my country promulgated the "Administrative Measures for the Labeling of Agricultural GMOs", which stipulates that the sales of agricultural GMOs listed in the catalog in China should have obvious labels. The new "Food Safety Law" implemented in October this year has further stipulated that genetically modified foods must be "conspicuously labeled", which will inevit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6895
Inventor 魏海燕张锡全汪万春曾静徐蕾蕊马丹魏咏新
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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