RPA (Recombinase Polymerase Amplification) detection method for CrylAb/CrylAc insect-resistant gene

A gene, insect-resistant plant technology, applied in DNA/RNA fragments, biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of not being able to further meet the rapid detection of genetically modified products

Active Publication Date: 2014-11-05
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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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, there is no gene-specific identification of Cry1Ab / Cry1Ac transgenic plants using RPA technology at home and abroad

Method used

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  • RPA (Recombinase Polymerase Amplification) detection method for CrylAb/CrylAc insect-resistant gene
  • RPA (Recombinase Polymerase Amplification) detection method for CrylAb/CrylAc insect-resistant gene
  • RPA (Recombinase Polymerase Amplification) detection method for CrylAb/CrylAc insect-resistant gene

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Embodiment Construction

[0012] 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.

[0013] 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.

[0014] First, design primers: according to TT51-1 (Cry1Ab / c, GenBank No. EU880444), Mon531 (Cry1Ac, GenBank No. AR656168), Bt11 (Cry1Ab, GenBank No. GV597352) and Mon15985 (Cry1Ac, GenBank No. EA135632) four Compare the different insect resistance gene sequences contained in different materials and design universal primers and probes in the conserved regions. RPA requires a pri...

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Abstract

The invention discloses a primer and probe combination applicable to recombinase polymerase amplification (RPA) and used for identifying insect-resistant plants containing a CrylAb / CrylAc transgene (a CrylAb gene, a CrylAc gene or a CrylAb / c gene). The sequence of a forward primer is represented as SEQ ID No.1, the sequence of a backward primer is represented as SEQ ID No.2 and the sequence of a probe is represented as SEQ ID No.3. Meanwhile, the invention further discloses a method for identifying plants modified with the gene; the method comprises the following steps: taking a DNA (Deoxyribose Nucleic Acid) of a sample to be detected as a template and carrying out RPA rapid amplification and real-time fluorescence detection by utilizing the primer; if an obvious amplification curve is obtained, showing that the DNA of the detected sample contains CrylAb / CrylAc transgene components. The invention provides an RPA detection method for gene specificity of CrylAb / CrylAc transgene insect-resistant plants.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to rapid identification of Cry1Ab / Cry1Ac genes (Cry1Ab gene, Cry1Ac gene or Cry1Ab / c fusion gene) component. 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 organisms and based on the principle of recombinase-polymerase-mediated amplificat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 金芜军宛煜嵩徐潮张秀杰苗朝华黄卫红
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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