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Qualitative PCR (polymerase chain reaction) detection primer for cry1A gene, detection method and detection kit

A detection method and a technology for detecting primers, which are applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., to achieve the effect of efficient technical means and wide applicability

Inactive Publication Date: 2018-04-03
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

However, since the developers of transgenic crops often carry out sequence modification or codon modification on the cry1A gene, the same type of cry1A gene in different transformants also has a large variation in the nucleotide sequence. Therefore, the establishment of a widely applicable The qualitative PCR detection method of cry1A gene provides an efficient technical means for the screening and detection of genetically modified crops, and is of great significance for improving the detection methods of genetically modified crops in my country

Method used

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  • Qualitative PCR (polymerase chain reaction) detection primer for cry1A gene, detection method and detection kit
  • Qualitative PCR (polymerase chain reaction) detection primer for cry1A gene, detection method and detection kit
  • Qualitative PCR (polymerase chain reaction) detection primer for cry1A gene, detection method and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Extraction and Detection of Plant Genomic DNA

[0047] Plant Genomic DNA Extraction Kit DP305 (Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to extract and purify DNA from plant materials. The extraction method was as follows:

[0048] 1) Take 200mg powder samples respectively;

[0049] 2) Add 800 μL of 65°C preheated GP1 buffer, quickly invert and mix well, place the centrifuge tube in a 65°C water bath for 1 hour, and invert the centrifuge tube to mix the samples several times during the water bath.

[0050] 3) Add an equal volume of phenol:chloroform (1:1), mix well, and centrifuge at 12000rpm for 10min.

[0051] 4) Transfer the supernatant to a new centrifuge tube, add an equal volume of chloroform, mix thoroughly, and centrifuge at 12000 rpm for 10 min.

[0052] 5) Take the supernatant, add an equal volume of GP2, and mix well.

[0053] 6) Transfer the mixed liquid into the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, and ...

Embodiment 2

[0060] Example 2 Sequencing verification of cry1A gene

[0061] Sequence comparison: By querying network databases, domestic and foreign patents, scientific papers, etc., the cry1A gene sequences in 11 different transgenic crops were collected, and DNAMAN software was used for sequence comparison analysis, and primers were designed to amplify the cry1A gene partial sequence. increase. See Table 1 for information on primers and transgenic materials.

[0062] Table 1 Information table of primers and transgenic materials

[0063]

[0064]

[0065] The cry1A gene in the 11 kinds of transgenic materials in Table 1 was amplified, and the qualitative PCR reaction system was as follows: 25 μL of Green Master Mix buffer, 2 μL of 10 μmol / L upstream primer, 2 μL of 10 μmol / L downstream primer, 2 μL of DNA template, make up to 50 μL with deionized water; the conditions of qualitative PCR reaction are: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds , ...

Embodiment 3

[0066] Example 3 Establishment of cry1A gene detection method

[0067] (1) Sequence comparison and primer design: DNAMAN software was used to compare and analyze the sequences obtained by sequencing, and according to the comparison results ( figure 1 ), the cry1A gene was divided into 4 categories, namely ①Bt11, Kefeng6, TT51-1, MON531, MON15985, MON88017; ②MON810, MON89034; ③Bt176, SK12-5; ④DAS-81419-2.

[0068] Primers were designed using Primer premier 5.0 software for the relatively conserved parts of the four types of sequences. A pair of cry1A gene qualitative PCR detection primers were screened out by using the degenerate primer strategy. The information of the degenerate primers is shown in Table 2. figure 2 .

[0069] Table 2 Degenerate primer information

[0070]

[0071] (2) Optimization of the PCR reaction system and reaction program: the final primer concentration was set to 4 gradient treatments of 0.1 μmol / L, 0.2 μmol / L, 0.4 μmol / L, and 0.8 μmol / L; the an...

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Abstract

The invention provides a qualitative PCR (polymerase chain reaction) detection primer for a cry1A gene, a detection method and a detection kit, and belongs to the technical field of biology. A detection primer pair comprises cry1A-aF and cry1A-aR; a nucleotide sequence of the cry1A-aF is shown in SEQ ID No.1, and a nucleotide sequence of the cry1A-aR is shown in SEQ ID No.2. The detection method and the detection kit can be used for detecting transgenic products containing the cry1A gene. The qualitative PCR detection primer for the cry1A gene has the advantages that the suitability is wide, and the sequence for modifying the sequence of the cry1A gene or modifying a codon can be accurately detected; a high-efficiency technical method is provided for the screening and detection of transgenic crops, and the important meaning on improving the transgenic crop detection method in China is realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a cry1A gene qualitative PCR detection primer, a detection method and a detection kit. Background technique [0002] Cry1A gene is a general term for a class of insect-resistant genes, and it is also the most common exogenous target gene in insect-resistant transgenic crops currently commercialized, including cry1Ab, cry1Ac, cry1A.105, mcry1Ac, cry1Ab / Ac, etc., cry1A gene in transgenic It can be used as a broadly representative target in detection work. However, since the developers of transgenic crops often carry out sequence modification or codon modification on the cry1A gene, the same type of cry1A gene in different transformants also has a large variation in the nucleotide sequence. Therefore, the establishment of a widely applicable The qualitative PCR detection method of cry1A gene provides an efficient technical means for the screening and detection of transgenic ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2600/158
Inventor 李亮董美宛煜嵩金芜军刘卫晓兰青阔温洪涛武利庆刘刚柳方方王迪
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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