Detection of CaMV 35S promoter and nos terminator by adopting RPA (Recombinase Ploymerase Amplification) technology

A P-35S, promoter technology, applied in recombinant DNA technology, DNA/RNA fragments, biochemical equipment and methods, etc., can solve the problems that cannot further meet the rapid detection of transgenic products, screening and identification of transgenic plants, etc.

Active Publication Date: 2014-01-15
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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Problems solved by technology

In the reported detection methods of genetically modified plants, the PCR instrument is mainly used for routine detection in the laboratory, and this method cannot further meet the rapid detection of genetically modified products.
At present, RPA technology has not been used to screen and identify transgenic plants at home and abroad

Method used

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  • Detection of CaMV 35S promoter and nos terminator by adopting RPA (Recombinase Ploymerase Amplification) technology
  • Detection of CaMV 35S promoter and nos terminator by adopting RPA (Recombinase Ploymerase Amplification) technology
  • Detection of CaMV 35S promoter and nos terminator by adopting RPA (Recombinase Ploymerase Amplification) technology

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Embodiment Construction

[0016] The present invention will be further clarified through the detailed description of specific embodiments below, but it is not intended to limit the present invention, but only for illustration.

[0017] For the experimental methods that do not indicate specific conditions in the following examples, generally follow conventional conditions, such as the conditions described in "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 2001) by Sambrook et al., or according to the conditions of the instrument Or the conditions recommended by the reagent manufacturer.

[0018] First, primer design and screening: The following factors are usually considered when designing primers: (1) GC content is 40%-60%; (2) try to avoid secondary structures inside the primer; (3) avoid repeated sequences in the primer . RPA requires a primer length of 30-35nt. RPA experiments need to design multiple pairs of primers from both ends of the target sequence for...

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Abstract

The invention provides an RPA sifting and detecting method for transgenic crops containing P-35S and T-nos elements, and particularly discloses a primer and probe combination applicable to the identification of transgenic plants containing transgenic elements, namely a CaMV 35S promoter and a nos terminator by adopting an RPA technology. The forward primer sequences of the primer and probe combination are shown in SEQ ID No.1 and SEQ ID No.4, the reverse primer sequences of the primer and probe combination are shown in SEQ ID No.2 and SEQ ID No.5, and the probe sequences of the primer and probe combination are shown in SEQ ID No.3 and SEQ ID No.6. In addition, the invention further discloses a method for identifying transgenic crops containing the two kinds of elements, and the method comprises the following steps: extracting the DNA of a sample to be detected as a template, and using the primer to perform RPA rapid amplification and real-time fluorescence detection, if an obvious amplification curve is obtained, the fact that the DNA of the detected sample contains P-35S and T-nos transgenic elements is proved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to rapid identification of CaMV 35S promoter (P-35S) and / or nos terminator (T- nos) components, specifically related to the combination of primers and probes and their monitoring methods. Background technique [0002] At present, DNA amplification is the main method of nucleic acid detection. The commonly used PCR detection requires sophisticated instruments and cumbersome test procedures, which is difficult to meet the requirements of on-site detection in non-laboratory environments. In recent years, nucleic acid constant temperature amplification technology has been greatly developed. Compared with traditional PCR, nucleic acid constant temperature amplification technology does not require expensive PCR equipment, and can quickly amplify the target fragment in a short time. It is simple, fast, sensitive, etc. advantage. RPA technology is developed by simulating DNA replication in orga...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q2521/101
Inventor 金芜军宛煜嵩徐潮苗朝华李亮黄卫红
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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