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Method for identifying transgenic Bt gene based on recombinase polymerase isothermal amplification method

A technology of recombinant enzyme polymerase and constant temperature amplification method, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc. It can solve the problems that PCR technology cannot implement rapid on-site detection, high operating technical requirements, and high instrument costs. , to achieve the effect of being conducive to on-site detection, low technical requirements, and good specificity

Pending Publication Date: 2022-01-11
JILIN AGRI SCI & TECH COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a series of detection methods based on PCR technology have some similar limitations, such as long detection time, complicated process, high instrument cost, and high requirements for operating techniques, etc., which limit the inability of PCR technology to be implemented quickly on-site. detection

Method used

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  • Method for identifying transgenic Bt gene based on recombinase polymerase isothermal amplification method
  • Method for identifying transgenic Bt gene based on recombinase polymerase isothermal amplification method
  • Method for identifying transgenic Bt gene based on recombinase polymerase isothermal amplification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] (1) Extraction of corn genomic DNA:

[0034] 1) Take 0.4 g of corn leaves from which the stems have been removed, put them into a pre-cooled mortar with liquid nitrogen added (the amount of liquid nitrogen added accounts for 2 / 3 of the volume of the mortar) and grind, pass through a 80-mesh sieve, Load into a 5mL centrifuge tube before the temperature rises;

[0035] 2) Add the improved 2×CTAB extract solution (0.1g / 100mL Tris-HCL, 1mL / 100mL β-mercaptoethanol, 3g / 100mL CTAB, 60mmol / L EDTA, 1.2mol / L NaCl) preheated at 65°C and add 0.04g PVP ) 1.6mL, incubate at 65°C, shake once every 5min, for a total of 30min;

[0036] 3) After the incubation, take it out, set the centrifuge at 4°C, 12000rpm, put the centrifuge tube into it carefully to avoid DNA damage, and centrifuge for 5min;

[0037] 4) Aspirate 1.4 mL of the supernatant with a micropipette, add a mixture of chloroform and isoamyl alcohol equal to the volume of the supernatant (wherein the volume of chloroform and...

Embodiment 2

[0052] (1) Extraction of corn genomic DNA:

[0053] 1) Take 0.4 g of corn leaves from which the stems have been removed, put them into a pre-cooled mortar with liquid nitrogen added (the amount of liquid nitrogen added accounts for 2 / 3 of the volume of the mortar) and grind, pass through a 90-mesh sieve, Load into a 5mL centrifuge tube before the temperature rises;

[0054] 2) Add the improved 2×CTAB extract solution (0.1g / 100mL Tris-HCL, 1mL / 100mL β-mercaptoethanol, 3g / 100mL CTAB, 60mmol / L EDTA, 1.2mol / L NaCl) preheated at 65°C and add 0.04g PVP ) 1.6mL, incubate at 65°C, shake once every 5min, for a total of 30min;

[0055] 3) Take it out after the incubation, set the centrifuge at 4°C, 12000rpm, put the centrifuge tube into it carefully to avoid DNA damage, and centrifuge for 8 minutes;

[0056] 4) Aspirate 1.4 mL of the supernatant with a micropipette, add a mixture of chloroform and isoamyl alcohol equal to the volume of the supernatant (wherein the volume of chloroform...

Embodiment 3

[0071] (1) Extraction of corn genomic DNA:

[0072] 1) Take 0.4 g of corn leaves from which the stems have been removed, put them into a pre-cooled mortar with liquid nitrogen added (the amount of liquid nitrogen added accounts for 2 / 3 of the volume of the mortar) and grind, pass through a 100-mesh sieve, Load into a 5mL centrifuge tube before the temperature rises;

[0073] 2) Add the improved 2×CTAB extract solution (0.1g / 100mL Tris-HCL, 1mL / 100mL β-mercaptoethanol, 3g / 100mL CTAB, 60mmol / L EDTA, 1.2mol / L NaCl) preheated at 65°C and add 0.04g PVP ) 1.6mL, incubate at 65°C, shake once every 5min, for a total of 30min;

[0074] 3) Take it out after the incubation, set the centrifuge at 4°C, 12000rpm, put the centrifuge tube into it carefully to avoid DNA damage, and centrifuge for 10min;

[0075] 4) Aspirate 1.4 mL of the supernatant with a micropipette, add a mixture of chloroform and isoamyl alcohol equal to the volume of the supernatant (wherein the volume of chloroform an...

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Abstract

The invention belongs to the technical field of transgenic crop identification, and provides a method for identifying a transgenic Bt gene based on a recombinase polymerase isothermal amplification method. The genomic DNA of a sample to be identified is extracted by using a CTAB method, the obtained genomic DNA of the sample to be identified is taken as a template, the primer is designed for RPA amplification reaction, the obtained amplification product is relatively high in purity and good in specificity, and the sensitivity reaches 6fg / mu L. Compared with a PCR technology commonly used in the market, the method has the advantages that the reaction time is shortened, the reaction temperature is reduced, the technical requirements on operators are relatively low, field detection is facilitated, and a new inspection idea is provided for inspection and quarantine departments.

Description

technical field [0001] The invention relates to the technical field of identification of transgenic crops, in particular to a method for identifying transgenic Bt genes based on a recombinase polymerase constant temperature amplification method. Background technique [0002] With the commercialization of the world's first transgenic maize Bt176 in 1996, transgenic maize has been widely planted around the world, with a total of 750 million hectares planted in 23 years (ISAAA, 2019). At present, the Bt gene has been widely used in transgenic crops. The protein encoded by this gene will cause insects to die soon after being ingested by insects, but it is harmless to humans. Because of its obvious effect on corn borer, cotton bollworm and armyworm, transgenic Bt corn has become one of the fastest commercially planted transgenic crops. [0003] Polymerase chain reaction technology (Polymerase chain reaction, PCR) is the main means of research in molecular biology, and is now wid...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12Q1/6895
CPCC12Q1/6844C12Q1/6895C12Q2600/16C12Q2521/507C12Q2521/119C12Q2522/101C12Q2527/101
Inventor 徐亚维郑雪刘相国陈子奇赵雪李鹏
Owner JILIN AGRI SCI & TECH COLLEGE
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