Primer group and kit for detecting Roundup Ready transgenic soybeans

A technology of genetically modified soybean and detection kit, which is applied in the direction of DNA/RNA fragment, recombinant DNA technology, microbial determination/inspection, etc. High yield and efficient amplification

Active Publication Date: 2012-02-01
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, referred to as LAMP) has many advantages. and there is currently no useful loop-mediated isothermal amplification technology for the detection of transgenic soybean rapid diagnostic kits

Method used

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  • Primer group and kit for detecting Roundup Ready transgenic soybeans
  • Primer group and kit for detecting Roundup Ready transgenic soybeans
  • Primer group and kit for detecting Roundup Ready transgenic soybeans

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Preparation of Example 1 Roundup Ready Transgenic Soybean Rapid Diagnostic Kit

[0073] (1) Synthesize EPSPS-NOS gene primers by DNA synthesizer according to the following sequence:

[0074] Outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO:1;

[0075] Outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO:2;

[0076] Inner primer FIP, its nucleotide sequence is shown in SEQ ID NO:3;

[0077] Internal primer BIP, its nucleotide sequence is shown in SEQ ID NO:4.

[0078] (2) Synthesize lectin gene primers by DNA synthesizer according to the following sequence:

[0079] Outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO: 19;

[0080] Outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO:20;

[0081] Internal primer FIP, its nucleotide sequence is shown in SEQ ID NO:21;

[0082] Internal primer BIP, its nucleotide sequence is shown in SEQ ID NO:22.

[0083] (3) Purchasing DNA polymerase:...

Embodiment 2

[0095] Example 2 Preparation of Roundup ready transgenic soybean rapid diagnostic kit

[0096] The two pairs of primers for the EPSPS-NOS gene are:

[0097] Outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO:5;

[0098] Outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO:6;

[0099] Inner primer FIP, its nucleotide sequence is shown in SEQ ID NO:7;

[0100] Internal primer BIP, its nucleotide sequence is shown in SEQ ID NO:8.

[0101] The two pairs of primers for the Lectin gene are:

[0102] Outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO:23;

[0103] Outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO:24;

[0104] Inner primer FIP, its nucleotide sequence is shown in SEQ ID NO:25;

[0105] Internal primer BIP, its nucleotide sequence is shown in SEQ ID NO:26.

[0106] The formula of the reaction solution is: each 1L reaction solution contains 1.6mmol dNTP, 20mmol Tris-HCl, 10mmol po...

Embodiment 3

[0109] Example 3 Preparation of Roundup ready transgenic soybean rapid diagnostic kit

[0110] The two pairs of primers for the EPSPS-NOS gene are:

[0111] The nucleotide sequence of the outer primer F3 is shown in SEQ ID NO:9;

[0112] The nucleotide sequence of the outer primer B3 is shown in SEQ ID NO:2;

[0113] The nucleotide sequence of the inner primer FIP is shown in SEQ ID NO:10;

[0114] The nucleotide sequence of the inner primer BIP is shown in SEQ ID NO:4;

[0115] The two pairs of primers for the Lectin gene are:

[0116] Outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO:23;

[0117] Outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO:24;

[0118] Inner primer FIP, its nucleotide sequence is shown in SEQ ID NO:25;

[0119] Internal primer BIP, its nucleotide sequence is shown in SEQ ID NO:26.

[0120] The formula of the reaction solution is: 2mmol dNTP, 25mmol Tris-HCl, 11mmol potassium chloride, 12mmol ammonium s...

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PUM

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Abstract

The invention discloses a primer group and a kit for detecting Roundup Ready transgenic soybeans. The kit consists of the primer group, Bst deoxyribonucleic acid (DNA) polymerase, a stabilizing solution, a reaction solution, a developing solution and a positive control solution. In the kit, six segments and four primers are utilized; and the kit has high specificity according to the condition that whether the existence of target materials can be judged or not through amplification. The quick diagnostic kit provided by the invention has high speed, high efficiency and high sensitivity, can be subjected to an amplification reaction only by a constant temperature without the usage of special reagents and equipment, has low detection cost and is easy and convenient to identify; pyrophosphate ions precipitated from deoxyribonucleoside triphosphate (dNTP) is combined with Mg<2+> in the reaction solution; the produced byproduct, namely magnesium pyrophosphate is precipitated and can be observed and identified through naked eyes; and after the developing solution is added, the kit has obvious development differences of negative and positive results and is more obvious and reliable. A soybean endogenous gene lectin is used as an internal label, so that errors and false negatives caused by the extraction of nucleic acid are prevented; and real-time monitoring is performed, so that detection time is greatly reduced, and human power and material resources are saved.

Description

technical field [0001] The invention relates to a biological detection reagent, in particular to a roundup ready transgenic soybean detection primer set and a diagnostic kit thereof. Background technique [0002] The glyphosate-resistant soybean variety developed by Monsanto in 1994 transformed the Agrobacterium tumefaciens The gene encoding 5-enolpyruvate shikimate-3-phosphate synthase (CP4 EPSPS) can resist the inhibition of the herbicide glyphosate on the shikimate synthesis pathway, allowing transgenic soybeans to grow normally after spraying the herbicide. The recipient of this variety is soybean variety A5403, which integrates exogenous CP4 EPSPS gene, E35S promoter and NOS terminator into 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 the supervision and management of genetically modified...

Claims

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Application Information

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