Method for identifying transgenic maize Bt176 strain specificity by RPA (recombinase polymerase amplification) technology

A transgenic corn, specific technology, applied in the direction of recombinant DNA technology, DNA / RNA fragments, microbial determination / inspection, etc., to achieve high sensitivity results

Inactive Publication Date: 2017-06-06
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Among the reported detection methods for transgenic maize BT176, the PCR instrument is mainly used for routine detection in the laboratory, and the loop-mediated isothermal amplification (LAMP) technology is also used for detection. At present, no RPA technology has been used. gene specific identification

Method used

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  • Method for identifying transgenic maize Bt176 strain specificity by RPA (recombinase polymerase amplification) technology
  • Method for identifying transgenic maize Bt176 strain specificity by RPA (recombinase polymerase amplification) technology
  • Method for identifying transgenic maize Bt176 strain specificity by RPA (recombinase polymerase amplification) technology

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Experimental program
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Embodiment 1

[0018] Firstly, primers and probes were designed according to the specific regions of transgenic maize BT176 transformants. The length of the primer is about 35nt. RPA experiments need to design multiple pairs of primers from both ends of the target sequence for optimization and screening. The increase, decrease or substitution of individual bases will have an important impact on the experimental results. A large number of primers were designed in this experiment, and a pair of primers with high sensitivity and specificity were screened out for RPA fluorescence detection. The sequences of primers and probes are listed in Table 1.

[0019] Table 1

[0020]

[0021] Note: FAM: luminescent group; dSpacer: abasic site; BHQ1: quenching group; block: closed group, carbonation used this time.

[0022] 1. Experimental materials

[0023] 1.1 Plant material

[0024] Genetically modified corn BT176 standard substance, genetically modified corn MON810 standard substance, geneticall...

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Abstract

The invention discloses a method for identifying the transgenic maize Bt176 strain specificity by a RPA (recombinase polymerase amplification) technology. The method is characterized in that the DNA (deoxyribonucleic acid) of a sample to be tested is firstly extracted as a template; screened primers and probes are used for performing RPA fast amplification and real-time fluorescence detection; if an amplification curve is obtained, the condition that the tested sample contains curve ingredients is proved. A large number of RPA primers are designed according to a connecting region of an exogenous inserted DNA sequence and a maize genome, a pair of primers and probe combinations capable of fast and effectively detecting the transgenic maize Bt176 ingredients are screened; a RPA detecting method of the transgenic maize Bt176 strain specificity is provided for the first time.

Description

technical field [0001] The invention relates to a strain identification method, in particular to a specific identification of transgenic maize Bt176 strain by using RPA technology. Background technique [0002] Nucleic acid isothermal amplification technology (isothermal nucleic acid amplification technology) is a new type of amplification technology, which can react under constant temperature conditions. It has the advantages of simplicity, speed, sensitivity, etc., and can be used for on-site inspection. RPA technology simulates the DNA replication process in organisms. The recombinase protein combines with single-stranded DNA (primer) under the participation of ATP to form DNA nucleoprotein filaments. The microfilament pulls the surrounding DNA molecules, compares the template DNA sequence and searches for a matching sequence, with the help of the single-strand binding protein, the double-stranded template DNA unwinds, and the primer and template pair to form a replicatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2521/507C12Q2522/101C12Q2563/107
Inventor 宛煜嵩李凯李亮董美金芜军刘卫晓温洪涛潘海会王永高进王迪
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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