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Loop-mediated isothermal amplification (LAMP) primer, kit and application of kit for T25 strain of transgenic maize

A transgenic corn and loop-mediated isothermal technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of detection of genetically modified ingredients in foods that have not been discovered by the LAMP method, and achieve simple operation, The effect of good application prospects

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

AI Technical Summary

Problems solved by technology

[0006] 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, kit and application of kit for T25 strain of transgenic maize
  • Loop-mediated isothermal amplification (LAMP) primer, kit and application of kit for T25 strain of transgenic maize
  • Loop-mediated isothermal amplification (LAMP) primer, kit and application of kit for T25 strain of transgenic maize

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] (1) Establishment of LAMP reaction system and primer screening

[0056] Use the standard sample of the T25 transgenic corn line with a concentration of 100% to carry out refined DNA extraction. For the experimental method of refined DNA, refer to the "Molecular Cloning Experiment Guide Third Edition" (written by Sam Brooke D.W Russell. Science Press 2002 [USA] ] J. Huang Peitang etc. translation).

[0057] Determination of concentration and purity of refined DNA:

[0058] 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:

[0059] C=A×N×50 / 1000 where:

[0060] C——DNA concentration (μg / μL)

[0061] A——absorbance value at 260nm

[0062] N——Nucleic acid dilution multiple

[0063] 1OD 260nm =50 μg / mL double-stranded DNA, when OD 260 / OD 280 When the ratio i...

Embodiment 2

[0074] The sensitivity of embodiment 2 LAMP primer T25

[0075] In order to detect the sensitivity of LAMP primer T25, the reaction time was determined according to the minimum sensitivity peak time and the earliest false positive peak time. Repeat the experimental method of Example 1, but the template and reaction conditions of LAMP T25 are adjusted as follows:

[0076] ①The template is as follows: Dilute the refined DNA of transgenic corn line T25 (100%, 100ng / μl) with non-transgenic corn AOCS0406-A DNA to 10%, 5%, 1%, 0.5%, 0.1%, 0.05% respectively Gradient, each dilution took 2μl for LAMP experiment; at the same time, with ddH 2 O was used as a negative control instead of the DNA template; then the refined DNA (100%, 100ng / μl) of the transgenic maize line T25 was used as a positive control to carry out LAMP amplification respectively; the results were detected by real-time turbidimetry;

[0077] 2. The constant temperature reaction time in the reaction conditions of embo...

Embodiment 3

[0084] Example 3 Specificity of LAMP Primer T25 (Real-time Nephelometric Method)

[0085]For verifying the specificity of LAMP reaction, repeat embodiment 2, the template DNA of LAMP reaction among the embodiment 2 is changed into following sample's refined DNA, and grouping:

[0086] In group CH1, the template for the reaction is the positive control - T25 genomic DNA (100%, 100ng / μl);

[0087] The DNA template of the CH2 group was a negative control (ddH 2 O);

[0088] CH3~32 are the DNA of the following samples in order: rice, corn, wheat, peanut, almond, mung bean, walnut, carrot, chickpea, lupine, green bean, orange, white cloud bean, soybean, oat, tomato, genetically modified Corn MON810, MON863, MON89034, BT176, GA21, BT11, NK603, transgenic rape RT73, transgenic soybean MON88017, MON89788, DP305423, DP356043, GTS40-3-2, transgenic potato EH92-527-1.

[0089] Reaction system and reaction conditions are as embodiment 2, LAMP T25 primer specificity detection result is ...

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Abstract

The invention discloses a primer which is used in LAMP detection, suitable for specificity detection of the T25 strain of transgenic maize, and provided with a base sequence such as SEQ ID NO: 2-7. The LAMP detection technique is adopted and nephelometry or a development process is combined to establish a standard method suitable for the specificity detection of the T25 strain of the transgenic maize in food. The method is simple in operation; expensive instruments are not dependent under a constant temperature condition; the detection time is about 1 hour, the method sensitivity reaches 0.5%, and the detection efficiency can reach that of existing polymerase chain reaction (PCR) method or fluorescence quantitative PCR method; the result is simple to judge, and the detection cost can reduced greatly; and the method can be applied to practical inspection and quarantine work, particularly to preliminary screening of mass samples and food quality assurance in various basic level laboratories, and can also serve as beneficial supplement of the existing PCR method.

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 T25 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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IPC IPC(8): C12Q1/68C12N15/11
Inventor 徐君怡曹际娟齐震玉姜丽
Owner 徐君怡
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