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Primer for detecting CAMV35S genes, relevant kit and detecting method

A gene detection and kit technology, applied in the field of inspection and detection, achieves the effects of significant color difference, high yield, and low detection cost

Active Publication Date: 2011-08-03
GUANGZHOU HUAFENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, isothermal amplification (Isothermal Amplification) nucleic acid rapid detection technology is a great progress in the detection technology of genetically modified products. The established loop-mediated isothermal amplification technology (loop-mediated isothermal amplification of DNA, referred to as LAMP) has many advantages. There is no useful loop-mediated isothermal amplification technology to detect CAMV35S gene rapid diagnostic kit in transgenic crops

Method used

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  • Primer for detecting CAMV35S genes, relevant kit and detecting method
  • Primer for detecting CAMV35S genes, relevant kit and detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Preparation of Transgenic Crop CAMV35S Gene Rapid Diagnostic Kit

[0041] (1) Synthesize oligodeoxynucleic acid primers by DNA synthesizer according to the following sequence:

[0042] Outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO: 1 or 5;

[0043]Outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO: 2 or 6;

[0044] Internal primer FIP, its nucleotide sequence is shown in SEQ ID NO:3 or 7;

[0045] Internal primer BIP, its nucleotide sequence is shown in SEQ ID NO:4 or 8.

[0046] (2) Purchasing DNA polymerase: Bst DNA polymerase (large fragment) in container.

[0047] (3) Prepare the reaction solution: The formula of the reaction solution contains 2mmol dNTP, 25mmol Tris-Cl, 12.5mmol potassium chloride, 12.5mmol ammonium sulfate, 10mmol magnesium sulfate, 1.25ml TritonX-100, 1mol betaine, 2 mol each of primers FIP / BIP and 0.25 mol each of outer primers F3 / B3 were prepared and placed in containers.

[004...

Embodiment 2

[0058] Example 2 Application of Transgenic Crop CAMV35S Gene Rapid Diagnostic Kit

[0059] In this example, the CAMV35S gene rapid diagnosis kit for transgenic crops prepared in Example 1 is used for the rapid diagnosis of the CAMV35S gene in the sample to be tested.

[0060] 1. Sample processing (template DNA extraction)

[0061] 1) Grind 100mg of the pretreated sample fully into powder in liquid nitrogen and add 700μL CTAB extraction buffer (the sample that does not need to be ground is directly added), shaken and mixed, and kept at 65°C for 30 minutes, during which time, gently invert and mix 2-3 times.

[0062] 2) Add 700 μ L of chloroform-isoamyl alcohol, and gently invert the solution 2-3 times. Centrifuge at 12000g for 5 min to separate the phases.

[0063] 3) Transfer the supernatant to a clean centrifuge tube, add 0.6 times the volume of 4°C pre-cooled isopropanol, let stand at -20°C for 5 minutes, and centrifuge at 12 000 g for 5 minutes.

[0064] 4) Discard t...

Embodiment 3

[0074] Example 3 Specificity Verification of Transgenic Crop CAMV35S Gene Rapid Diagnostic Kit

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Abstract

The invention discloses a primer for detecting CAMV35S genes, a relevant kit and a detecting method. The primer for detecting the CAMV35S genes comprises an outer primer F3, an outer primer B3, an inner primer FIP and an inner primer BIP, and the nucleotide sequences of the primer are respectively shown as SEQ ID NO.1-4. The primer can judge whether target matter exists or not according to amplification, and has high specificity. The kit in the invention can rapidly diagnose the CAMV35S genes, can amplify at only a constant temperature without special reagents or equipment, has low detection cost and is suitable for popularization and application.

Description

technical field [0001] The invention relates to the field of inspection and detection, in particular to a CAMV35S gene detection primer, a corresponding kit and a detection method. Background technique [0002] CAMV35S gene is the 35S promoter of cauliflower mosaic virus, which is widely used in transgenic crops such as transgenic soybean, corn, rape, potato and tomato. [0003] In order to strengthen the supervision and management of genetically modified products, a variety of genetically modified component detection methods have been developed, from protein-based ELISA detection technology to nucleic acid-based polymerase chain reaction (PCR) technology and the rapidly developing gene chip technology. Among them, PCR detection technology is the most general because of its sensitivity, specificity, and high efficiency. This technology is identified by detecting exogenous nucleic acid fragments contained in genetically modified products. The emergence of GM technology not o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 曹以诚杜正平李志勇高东微陈洵谭慧媚
Owner GUANGZHOU HUAFENG BIOTECH
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