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Loop-mediated isothermal amplification (LAMP) primer of genetically modified maize BT11 strain, reagent kit and application of LAMP primer

A genetically modified corn, ring-mediated isothermal technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems such as the detection of genetically modified ingredients in foods that have not yet been found by the LAMP method, and achieve simple results. , The effect of reducing inspection costs and saving time

Inactive Publication Date: 2012-12-19
徐君怡 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] No standardized research on the application of LAMP method to the detection of food genetically modified ingredients has been found so far

Method used

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  • Loop-mediated isothermal amplification (LAMP) primer of genetically modified maize BT11 strain, reagent kit and application of LAMP primer
  • Loop-mediated isothermal amplification (LAMP) primer of genetically modified maize BT11 strain, reagent kit and application of LAMP primer
  • Loop-mediated isothermal amplification (LAMP) primer of genetically modified maize BT11 strain, reagent kit and application of LAMP primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] (1) Establishment of LAMP reaction system and primer screening

[0052] The mixture composed of transgenic maize BT11 and non-transgenic maize AOCS0406-A with a mass ratio of 5:95 is called the standard sample of BT11 transgenic maize line with a concentration of 5%; the DNA is refined. For the experimental method of DNA extraction, refer to "Molecular Cloning Experiment Guide, Third Edition" (written by Sambrook D.W Russell. Science Press, 2002 [US] J. Huang Peitang, etc. translation).

[0053] Wherein, the determination of the concentration and purity of the above-mentioned finely extracted DNA:

[0054] Take 5 μL DNA solution and add ddH 2 O gradient diluted to 1mL, using a nucleic acid protein analyzer or ultraviolet spectrophotometer to measure the optical density at 260nm and 280nm. The concentration of DNA was calculated according to the following formula:

[0055] C=A×N×50 / 1000 where:

[0056] C——DNA concentration (μg / μL)

[0057] A——absorbance value at 260...

Embodiment 2

[0068] Embodiment 2 LAMP primer specificity experiment

[0069] Separately extract the DNA of the following samples:

[0070] Transgenic corn line BT11 and peas, lentils, rice, lupins, potatoes, corn, soybeans, crabs, mung beans, walnuts, carrots, wheat, genetically modified rice BT63, Kefeng 6, Kefeng 8, genetically modified corn BT176, EVENT98140, GA21, MON863, MON810, MIR604, MON88017, MON89034, transgenic potato EH92-527-1, transgenic soybean A5547-127, GTS40-3-2, DP305423, DP356043, MON89788, transgenic rapeseed GT73;

[0071] Each of the above-mentioned DNA samples that were extracted (after extraction, the concentration of the sample DNA was 100ng / μl) was used as the template of the LAMP reaction, and the BT11 primer designed in this experiment was used as the LAMP primer to perform the LAMP (real-time turbidity method) reaction respectively. :

[0072] The reaction system is the same as in Example 1, and the reaction conditions are: incubate at 63° C. for 60 minutes,...

Embodiment 3BT1

[0077] Embodiment 3BT11 primer sensitivity experiment

[0078] Dilute the BT11 DNA standard of the transgenic corn line with the DNA standard of non-transgenic corn AOCS0406-A (DNA concentration is 100ng / μl), and dilute the BT11 DNA standard of the transgenic corn line as a template to 5% respectively (the concentration of BT11 DNA is 5ng / μl , AOCS0406-A DNA concentration is 95ng / μl), 2% (wherein BT11DNA concentration is 2ng / μl, AOCS0406-A DNA concentration is 98ng / μl), 1% (wherein BT11DNA concentration is 1ng / μl, AOCS0406-A DNA concentration is 99ng / μl), 0.5% (the concentration of BT11DNA is 0.5ng / μl, the concentration of AOCS0406-A DNA is 99.5ng / μl), 0.1% (the concentration of BT11DNA is 0.1ng / μl, and the concentration of AOCS0406-A DNA is 99.9ng / μl ) several gradients, for the convenience of expression, hereinafter referred to as BT11DNA 5% (5ng / μl), BT11DNA 2% (2ng / μl), BT11DNA 1% (1ng / μl), BT11DNA 0.5% (0.5ng / μl) and BT11DNA 0.1 % (0.1ng / μl), take 2μl of each dilution fo...

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Abstract

The invention discloses a loop-mediated isothermal amplification (LAMP) primer of a genetically modified maize BT11 strain, a reagent kit and application of the LAMP primer. The primer used for the LAMP detection has base sequences being SEQ ID NO:2-7, the LAMP detection technology is adopted, a turbidity method or a color development method is combined, and a standard method applicable to the specificity detection of the genetically modified maize BT11 strain in the food is built. The method has the advantages that the operation is simple, the dependence on expensive instruments is avoided, the detection time is about one hour, the sensitivity of the method reaches 0.5 percent, the detection efficiency is the same as that of the traditional PCR (polymerase chain reaction) method or a fluorescent quantification PCR method, and the method can be applied to inspection and quarantine practical work, is particularly applied to the fast preliminary screening of a plurality of samples and the food quality monitoring of various kinds of basic laboratories and is suitable for being used as industrial recommended standards to be applied and popularized.

Description

technical field [0001] The invention belongs to the field of food inspection techniques and methods, and in particular relates to a loop-mediated isothermal amplification (LAMP) primer, a kit and a detection method for a transgenic corn BT11 strain in food. Background technique [0002] Corn is one of the most important food crops in the world, and its yield per unit area ranks first. There are more than 350 kinds of corn pests in the world, among which the Lepidoptera corn borer is the most widely distributed and the most harmful. It is an important corn pest worldwide and seriously affects the yield and quality of corn. Over the past ten years, breakthroughs have been made in the cultivation of transgenic plants, and a number of new varieties have been introduced, which have played an obvious role in improving crop production and have also shown great potential. However, there is much debate about the safety of GM corn. In the face of the impact of biogenetic engineering...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 徐君怡曹际娟郑秋月赵昕
Owner 徐君怡
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