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Reference molecule for specifically detecting genetically modified rapeseed RT73 and application of reference molecule

A transgenic, rapeseed technology, applied in the biological field, can solve problems such as affecting the stability of standard molecules

Active Publication Date: 2011-08-31
SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF P R C
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, the difficulty lies in the selection of a suitable carrier, such as the size of the carrier will affect the stability of the standard molecule

Method used

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  • Reference molecule for specifically detecting genetically modified rapeseed RT73 and application of reference molecule
  • Reference molecule for specifically detecting genetically modified rapeseed RT73 and application of reference molecule
  • Reference molecule for specifically detecting genetically modified rapeseed RT73 and application of reference molecule

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Embodiment 1, standard molecular construction

[0095] In order to construct a standard molecule suitable for the specific detection of transgenic rapeseed RT73 strains, the RT73' end adjacent sequence (316bp), rapeseed endogenous standard gene PEP (307bp) and HMG (311bp) three fragments were cloned into the pEASY-T3 vector, Get pEASY-RT73 ( figure 1 ), as a standard molecule, the size of the standard molecule is 3935bp. The results of three times of plasmid whole gene sequencing were consistent with the target sequence, indicating that the plasmid pEASY-RT73 was successfully constructed.

Embodiment 2

[0096] Embodiment 2, standard molecule is used for the applicability identification of ordinary PCR detection

[0097] (a) Specificity test

[0098] Nine kinds of transgenic rape strain-specific detection primers were used to test the specificity of standard molecules, and the results were as follows: figure 2 shown. In the amplification using standard molecular DNA as a template, obvious target bands can only be obtained in the amplification of the strain-specific sequence at the 3' end of RT73' and endogenous standard genes PEP and HMG ( figure 2 A).

[0099] There were no visible bands when amplified with transgenic rapeseed T45, Oxy235, MS8, MS1, RF1, RF2, RF3 and Topas19 / 2 strain-specific detection primers, but the positive controls corresponding to each detection system had target bands ( figure 2 B).

[0100] Therefore, the standard molecule pEASY-RT73 is highly specific for the detection of the transgenic rapeseed RT73 line.

[0101] (b) Detection sensitivity ...

Embodiment 3

[0103] Embodiment 3, standard molecule is used for the applicability identification of real-time fluorescent PCR detection

[0104] (a) Preparation of standard curve

[0105] Using gradiently diluted standard molecular DNA as a template, real-time fluorescent PCR amplification was performed on three target fragments (RT733' adjacent sequence, HMG, and PEP) to make a standard curve. like image 3 As shown, the slopes of the standard curves of the three target fragments were -3.27, -3.38 and -3.43, respectively, and the reaction efficiencies were 1.02, 0.98 and 0.96, R 2 All greater than 0.999.

[0106] Therefore, the standard curve is suitable for further quantitative analysis of the samples.

[0107] (b) Limit of detection (LOD) and limit of quantitation (LOQ) tests

[0108] Among the 25 amplifications using 50 copies of DNA as a template, RT73 strain-specific sequence, PEP and HMG three target fragments had 25 amplification curves, and the RSD values ​​were 2.04%, 3.06% a...

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Abstract

The invention relates to a reference molecule for specifically detecting a genetically modified rapeseed RT73 and application of the reference molecule. The reference molecule which is suitable for specific detection of a genetically modified rapeseed RT73 strain is constructed, and contains an exogenous interpose fragment of the genetically modified rapeseed RT73 strain, a 3' terminal adjacent area sequence of genomic deoxyribonucleic acid (DNA) of rape, and HMG and PEP fragments of an endogenous reference gene of the rape. The reference molecule has high specificity on the detection of the genetically modified rapeseed RT73 strain.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to a standard molecule for specifically detecting transgenic rapeseed RT73 and its application. Background technique [0002] While genetically modified crops solve the growing food crisis around the world, they also bring food safety and environmental safety problems. Therefore, governments around the world and relevant international organizations attach great importance to the supervision and management of genetically modified crops and their products. As one of the important technical supports for the supervision of genetically modified products, nucleic acid detection technology has become the most important and commonly used method for the detection of genetically modified organisms. Regardless of the method based on nucleic acid or protein, reference materials must be used as positive controls or standard curves for the detection of genetically modified...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12Q1/68
Inventor 李想潘良文吕蓉刘月明张舒亚高琴
Owner SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF P R C
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