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RPA primer for detecting transgenic corn Bt11 strain and detecting method

A technology of genetically modified corn and detection method, applied in the field of plant molecular biology, can solve the problem of difficult to meet the rapid screening and detection of genetically modified nucleic acid components, and achieve the effects of shortening the RPA reaction time, high amplification efficiency and good specificity

Inactive Publication Date: 2018-05-15
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PCR technology has been widely used in the detection of genetically modified genes. However, conventional PCR detection requires sophisticated thermal cyclers and complex experimental procedures, which is difficult to meet the needs of rapid screening and detection of genetically modified nucleic acid components in non-laboratory environments.

Method used

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  • RPA primer for detecting transgenic corn Bt11 strain and detecting method
  • RPA primer for detecting transgenic corn Bt11 strain and detecting method
  • RPA primer for detecting transgenic corn Bt11 strain and detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 A kind of detection transgenic maize Bt11 line RPA primer acquisition

[0039] 1. Design primers

[0040]Six pairs of RPA primers were designed according to the sequence of the exogenous gene phosphinothricinacetyl transferase gene (PAT) of transgenic maize Bt11 and the junction region of the maize genome. The specific sequences are shown in Table 1.

[0041] Table 1

[0042]

[0043] 2. Screening primers

[0044] 1) extract the DNA of transgenic corn Bt11, the method is as follows:

[0045] Add about 20 mg of transgenic corn Bt11 seed powder to the EP tube, then add Buffer GF1 (400 μL) and RNase A (4 μL) in the Kangwei (Kangwei Century Biotechnology Co., Ltd. (Beijing)) kit, and shake together for about 1 minute, 65°C water bath for 10 minutes, vortexed 2-3 times during the period; add 130 μL Buffer GF2, mix well, incubate on ice for 5 minutes; centrifuge at 14000rpm (~20000×g) for 5 minutes, attention: do not Mix Buffer GF1 with RNase A before use....

Embodiment 2

[0055] Example 2 Verification of a method for detecting transgenic maize Bt11 strains

[0056] 1. Specificity Verification

[0057] Genomic DNA of transgenic corn GA21 seed powder, Bt176 seed powder and NK603 seed powder was extracted with Kangwei Century Genomic DNA Extraction Kit (Kangwei Century Biotechnology Co., Ltd. (Beijing)), and the specific extraction experimental steps were the same as in Example 1.

[0058] The RPA reaction was carried out with the genomic DNA of transgenic maize Bt11, GA21, Bt176 and NK603 as template DNA and the RPA primer pair of the present invention as primers respectively.

[0059] Among them, the total system of RPA reaction is 50 μL, including: ddH 2 O 10.0 μL, Rehydration Buffer 29.5 μL, forward primer with a concentration of 10 μmol / L, reverse primer 3.0 μL each, template DNA with a concentration of 100 ng / μL 2.0 μL, magnesium acetate solution 2.5 μL that comes with the kit, kit Comes with freeze-dried enzyme powder.

[0060] The RPA r...

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Abstract

The invention discloses a RPA primer for detecting a transgenic corn Bt11 strain and a detecting method. The RPA primer is designed according to transgenic corn Bt11 exogenous gene phosphinothricin acetyltransferase gene PA and a sequence of corn genome connecting area; the RPA primer comprises a forward primer F and a reverse primer R, and the specific sequence is as follows: F: 5'-CTGGGAGGCCAAGGTATCTAATCAGCCATCCC-3'; R: 5'-CTGCTGTAGCTGGCCTAATCTCAACTGGTCTCC-3'; a quick detecting method based on a recombinase polymerase isothermal amplification technology is established for quickly identifyingthe transgenic corn Bt11 strain, so that the specificity is strong, the sensitivity is high, index amplification of a target segment within short time can be realized, requirements on environmental equipment are low, and operations are simple and convenient, and therefore, the RPA primer has a wide popularization and application prospect in the transgenic product component detecting field.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and in particular relates to an RPA primer and a detection method for detecting a transgenic corn Bt11 strain. Background technique [0002] With the rapid development of research and application of transgenic technology, research on detection technology of genetically modified products has become an important part of the global safety management of genetically modified organisms. PCR technology has been widely used in the detection of genetically modified genes. However, conventional PCR detection requires sophisticated thermal cyclers and complex experimental procedures, which is difficult to meet the needs of rapid screening and detection of genetically modified nucleic acid components in non-laboratory environments. [0003] In recent years, nucleic acid isothermal amplification technology has been developed rapidly. Compared with conventional PCR, nucleic acid isothermal amplification ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2521/507C12Q2522/101C12Q2531/119
Inventor 武国干唐雪明潘爱虎孙宇李鹏吴潇吕贝贝蒋玮王金斌
Owner SHANGHAI ACAD OF AGRI SCI
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