Primer group for testing Roundup Ready transgenic soy bean EPSPS gene, rapid diagnosis kit and testing method thereof

A technology for rapid diagnosis of transgenic soybeans, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of unseen detection of EPSPS gene rapid diagnostic kits for transgenic crops, and achieve significant color difference , high yield and low detection cost

Active Publication Date: 2012-10-03
GUANGZHOU HUAFENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, isothermal amplification (Isothermal Amplification) nucleic acid rapid detection technology is a great progress in the detection technology of genetically modified products. The established loop-mediated isothermal amplification technology (loop-mediated isothermal amplification of DNA, LAMP) has many advantages. and there is no useful loop-mediated isothermal amplification technology to detect transgenic crops EPSPS gene rapid diagnostic kit

Method used

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  • Primer group for testing Roundup Ready transgenic soy bean EPSPS gene, rapid diagnosis kit and testing method thereof
  • Primer group for testing Roundup Ready transgenic soy bean EPSPS gene, rapid diagnosis kit and testing method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0057] The preparation of embodiment 1 transgenic crop EPSPS gene rapid diagnosis kit

[0058] (1) Synthesize oligodeoxynucleic acid primers by DNA synthesizer according to the following sequence:

[0059] Outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO: 1;

[0060] Outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO: 2;

[0061] Internal primer FIP, the nucleotide sequence of which is shown in SEQ ID NO: 3;

[0062] The nucleotide sequence of the internal primer BIP is shown in SEQ ID NO:4.

[0063] (2) Purchase DNA polymerase: Bst DNApolymerase (large fragment) and place it in a container.

[0064] (3) Preparation of reaction solution: The formula of the reaction solution contains 2mmol dNTP, 25mmol Tris-Cl, 12.5mmol potassium chloride, 12.5mmol ammonium sulfate, 10mmol magnesium sulfate, 1.25ml TritonX-100, 1mol betaine, 2 mol each of primers FIP / BIP and 0.25 mol each of outer primers F3 / B3 were prepared and placed in containe...

Embodiment 2

[0075] The preparation of embodiment 2 transgenic crops EPSPS gene rapid diagnosis kit

[0076] The two pairs of primers are:

[0077] Outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO: 5;

[0078] Outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO: 6;

[0079] Internal primer FIP, the nucleotide sequence of which is shown in SEQ ID NO: 7;

[0080] The nucleotide sequence of the internal primer BIP is shown in SEQ ID NO:8.

[0081] The formula of the reaction solution is: each 1L reaction solution contains 1.6mmol dNTP, 20mmol Tris-HCl, 10mmol potassium chloride, 10mmol ammonium sulfate, 8mmol magnesium sulfate, 1ml TritonX-100, 0.8mol betaine, each of the internal primers FIP / BIP 1.6mol and 0.2mol each of outer primer F3 / B3;

[0082] The chromogenic solution is EvaGreen.

[0083] Others are the same as embodiment 1.

Embodiment 3

[0084] Example 3 Application of Transgenic Crops EPSPS Gene Rapid Diagnostic Kit

[0085] In this example, the EPSPS gene rapid diagnosis kit for transgenic crops prepared in Example 1 is used for rapid diagnosis of the EPSPS gene in the sample to be tested.

[0086] 1. Sample processing (template DNA extraction)

[0087] 1) Grind 100mg of the pretreated sample fully into powder in liquid nitrogen, then add 700 μL CTAB extraction buffer I (directly add the sample that does not need to be ground), shake and mix, and keep warm at 65°C for 30 minutes, during which Mix by gentle inversion 2-3 times from time to time.

[0088] 2) Add 700 μL of chloroform-isoamyl alcohol, and gently invert the solution 2-3 times. Centrifuge at 12000g for 5min to separate the phases.

[0089] 3) Transfer the supernatant to a clean centrifuge tube, add 0.6 times the volume of 4°C pre-cooled isopropanol, let stand at -20°C for 5 minutes, and centrifuge at 12000g for 5 minutes.

[0090] 4) Discard t...

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Abstract

The invention discloses a primer group for testing Roundup Ready transgenic soy bean EPSPS gene based on the loop-mediated isothermal amplification of DNA (LAMP), a rapid diagnosis kit and a testing method thereof. The kit comprises the primer group, Bst DNA polymerase, stabilizing solution, reacting solution, developing solution and positive control solution. The kit applies six sections and four primers, can judge whether the target substance is existent or not according to the amplification, so that the kit has a high specificity. The rapid diagnosis kit of the invention is rapid, efficient and sensitive; the amplification reaction can be realized only needing constant temperature without the special agent or equipment; the testing cost is low and the test process is simple. Pyrophosphate ions separated from the dNTP are combined with Mg<2> in the reaction solution to generate the byproduct, i.e. magnesium pyrophosphate deposition, which can be observed and appraised by viewing; and the developing differences of the negative and positive results are obviously by adding the developing solution, and the results are more obvious and reliable.

Description

technical field [0001] The invention relates to a biological detection reagent, in particular to a roundup ready transgenic soybean EPSPS gene detection primer set, a rapid diagnostic kit and a detection method. Background technique [0002] The glyphosate-resistant soybean variety developed by Monsanto in 1994 has transformed the gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (CP4EPSPS) from the microorganism Agrobacterium tumefaciens, which can resist the herbicide glyphosate. Inhibition of the shikimic acid synthesis pathway allowed transgenic soybeans to grow normally after spraying herbicides. The recipient of this variety is soybean variety A5403, and the exogenous CP4EPSPS gene, E35S promoter and NOS terminator are integrated in the transgenic soybean genome. This variety is currently the transgenic soybean variety with the largest planting area, and is widely used in the processing and production of edible oil and food. [0003] In order to strengthen th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 曹以诚杜正平陈洵李志勇高东微
Owner GUANGZHOU HUAFENG BIOTECH
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