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Sensor detecting method for neomycin phosphoric acid transferase gene in transgene plants

A technology of phosphotransferase and transgenic plants, which is applied in the field of sensor detection of neomycin phosphotransferase gene in transgenic plants, which can solve the problems of high cost and time-consuming detection

Active Publication Date: 2008-09-10
中谱安信(青岛)检测科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the existing detection methods are all based on PCR or fluorescent PCR. There is no use of loop-mediated isothermal amplification technology to detect NPT II gene, and there is no report of the use of loop-mediated isothermal amplification technology combined with nano-label electrochemical DNA sensor technology. The report on the detection of NPT II gene is used, and the existing methods have disadvantages such as high detection cost and long time-consuming

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Make the nano PbS-probe sequence labeling solution according to the following formula:

[0040] (1) Labeling of probe sequences by nano-PbS

[0041] Add 11.0 μL thioglycolic acid to 50.0 mL Pb(NO 3 ) 2 Mix 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 2 S solution 30.0mL (nitrogen protection). After the dropwise addition, the stirring was continued for 24h, and the mixture gradually turned brown. The PbS quantum dots prepared by this method have good stability.

[0042] Take 5.0mL PbS nano sol for centrifugal purification, wash with water and disperse in 2.0mL water, add 100μL 50.0mmol / L EDC, 100μL 50.0mmol / L NHS and 0.1mmol / L probe sequence, stir at room temperature for 18h, the reactant is heated at 10000r Centrifuge at 1 / min for 30 min, wash with PBS solution several ...

Embodiment 2

[0058] The following method was used to detect the exogenous gene NPT II in transgenic tomato (Zeneca) using the nano-lead sulfide-labeled electrochemical DNA sensing technique combined with loop-mediated isothermal amplification:

[0059] (1) LAMP reaction solution:

[0060] 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, 1U / μL UNGase and 4 μL ddH 2 O (sterilized double distilled water).

[0061] The upstream internal primer, downstream internal primer, upstream external primer, and downstream external primer are the same as above.

[0062] The mass ratio of the mixture of the four deoxyribonucleic acids in the dNTP is dUTP:dATP:dGTP:dCTP=2:1:1:1.

[0063] (2); Bst DNA polymerase: 8U / μL;

[0064] Follow t...

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Abstract

The invention relates to a sensor detecting method for neomycin phosphotransferase genes in transgene plants, which is characterized in that: the method comprises the following steps that: an amplification reactant namely a double-strand target sequence of neomycin phosphotransferase gene is obtained through loop-mediated isothermal amplification, the double-strand target sequence is pyrolyzed to single-strand target sequences in water bath, the single-strand target sequences are in self assembly fixture on the surface of a gold electrode, and a target sequence modified electrode of the neomycin phosphotransferase gene is obtained, and then nanometer lead sulfide is marked with a probe sequence from the neomycin phosphotransferase gene to obtain a marked probe sequence. The marked probe sequence and the target sequence on the target sequence modified electrode are performed molecular hybridization, and performed electrochemical detection by a coordinate mercury plating anodic stripping method to detect corresponding lead ions. The sensor detecting method for the neomycin phosphotransferase genes in transgene plants has the advantages of rapidness, strong specificity, high sensitivity and convenient use.

Description

technical field [0001] The invention relates to a method for detecting transgenic exogenous gene-neomycin phosphotransferase (NPT II) by using loop-mediated isothermal amplification (LAMP) technology combined with nano-label electrochemical DNA sensor technology, that is, in transgenic plants A sensor detection method for neomycin phosphotransferase gene. Background technique [0002] Phosphotransferase (Neomycin Phosphotransferase II, NPT II) gene is by far the most widely used selection marker in plant genetic transformation. The gene encodes neomycin phosphotransferase, also known as aminoglycoside-3'-phosphotransferase II, and its encoded product can make aminoglycoside antibiotics (aminoglycoside angibi-otics) such as kanamycin ( Kanamycin, Km), neomycin (neomycin), G418 and other phosphorylation and inactivation. Aminoglycoside antibiotics such as kanamycin can combine with the ribosomal 30S small subunit in plant cell chloroplasts and mitochondria, affect the format...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N27/327
Inventor 高宏伟孙伟孙敏刘彩霞焦奎
Owner 中谱安信(青岛)检测科技有限公司
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