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Micro-pore board fixing and nano lead sulfide marking electrochemistry detecting method for transgene soybean

A technology of transgenic soybeans and microplates, applied in biochemical equipment and methods, electrochemical variables of materials, measurement/inspection of microorganisms, etc., can solve the problems of time-consuming and high detection costs

Inactive Publication Date: 2008-09-10
QINGDAO UNIV OF SCI & TECH
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Problems solved by technology

However, the existing detection methods are all based on or utilize complex PCR or fluorescent PCR technology, and have not used loop-mediated isothermal amplification technology to detect CP4 EPSPS gene, nor used loop-mediated isothermal amplification technology and nano-label electrochemical A report on detection of CP4 EPSPS gene by combination of analytical techniques
Existing methods have disadvantages such as high detection cost and time-consuming

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Make the nano CdS-probe sequence labeling solution of transgenic soybean exogenous gene CP4 EPSPS probe sequence according to the following formula:

[0031] (1) Add 11.0μL thioglycolic acid to 50.0mL Pb(NO 3 ) 2Mix well in the solution, use 0.5mol / L NaOH solution to adjust the pH value of the mixed solution to about 7, pass nitrogen gas to remove oxygen for 30 minutes, slowly add 1.5mmol / L NaOH dropwise to the above mixed solution under magnetic stirring and nitrogen protection 2 S solution 30.0mL. After the dropwise addition, the stirring was continued for 24h, and the mixture gradually turned brown. The nano-PbS prepared by this method has good stability.

[0032] (2) Take 5.0 mL of nano-PbS sol for centrifugal purification, wash with water and disperse in 2.0 mL of water, add 100 μL 50.0 mmol / L EDC, 100 μL 50.0 mmol / L NHS and 0.1 mmol / L probe sequence, stir at room temperature for 18 h, the reaction The mixture was centrifuged at 10,000 r / min for 30 min, washed ...

Embodiment 2

[0045] 1. Make the loop-mediated isothermal amplification kit for detecting the exogenous gene CP4 EPSPS of transgenic soybean (line GTS 40-3-2) according to the following formula:

[0046] (1) LAMP reaction solution:

[0047] Contains 2.5 μL 10× Thermopol reaction buffer, 1.0 μL 10 mmol / L dNTP, 1.0 μL 20 μmol / L upstream internal primer (FIP), 1.0 μL 20 μmol / L downstream internal primer (BIP), 0.25 μL 20 μmol / L upstream external primer ( F3), 0.25 μL 20 μmol / L downstream outer primer (B3), 0.5 μL 100 mmol / L MgSO 4 , 12.5 μL 2mol / L betaine, UNG enzyme: 1 μL of 1U / μL and 4 μL ddH 2 O.

[0048] The upstream internal primer, downstream internal primer, upstream external primer, and downstream external primer are the same as above.

[0049] The mass ratio of the mixture of the four deoxyribonucleic acids in the dNTP is dUTP:dATP:dGTP:dCTP=2:1:1:1.

[0050] (2) Bst polymerase B: 8U / μL;

[0051] 2. Use GBT 19495.3-2004 method to extract DNA from the sample

[0052] 3. Transgeni...

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Abstract

The invention relates to a method for microplate fixing and nanometer lead sulfide marker electrochemical detecting genetically modified soybeans, which is characterized in that: the method comprises the following steps that: an amplification reactant namely a double-strand target sequence of an exogenous gene CP4 EPSPS to be detected is obtained through loop-mediated isothermal amplification, the reactant amplimer strain is exogenous gene CP4E PSPS of GTS 40-3-2 genetically modified soybeans, the double-strand target sequence is pyrolyzed to single-strand target sequences in water bath to fix on a microplate, and nanometer lead sulfide is led to marking a probe sequence from the exogenous gene CP4E PSPS to obtain a marked probe sequence, the marked probe sequence and the target sequence fixed on the microplate are performed molecular hybridization, and performed electrochemical detection with coordinate mercury plating anodic stripping voltammetry, aiming to electrochemically detect the exogenous gene (CP4 EPSPS) of the detected genetically modified soybeans. The method for microplate fixing and nanometer lead sulfide marker electrochemical detecting genetically modified soybeans has the advantages of rapidness, strong specificity, high sensitivity and convenient use.

Description

technical field [0001] The present invention relates to a method for rapid detection of genes, specifically, a method for detecting exogenous genes (CP4 EPSPS) in transgenic soybeans by combining loop-mediated isothermal amplification (LAMP) technology and nanometer lead sulfide marker gene sequence electrochemical analysis technology—micro Method of hole plate immobilization and nano-aluminum sulfide labeling for electrochemical detection of transgenic soybean. Background technique [0002] In recent years, the global planting area of ​​genetically modified plants has grown rapidly, and the leading genetically modified soybean accounts for more than 63% of the global genetically modified crops. All genetically modified soybeans are herbicide-resistant soybeans, that is, glyphosate-resistant genetically modified soybeans. The exogenous protein of this strain of soybean is CP4EPSPS, which can effectively degrade the herbicide glyphosate, and it has been proved by animal exper...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N27/26
Inventor 孙伟高宏伟焦奎钟江华覃鹏
Owner QINGDAO UNIV OF SCI & TECH
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