DNA standard sample for transgenic component detection and application of DNA standard sample

A technology for genetically modified components and standard samples, which is applied in recombinant DNA technology, DNA/RNA fragments, and microorganism determination/inspection, etc., which can solve problems such as increasing the workload of testing, pushing up testing costs, and inconvenient testing.

Inactive Publication Date: 2019-10-25
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the genetically modified screening test, according to the detected target, the genetically modified material containing the corresponding target should be selected as the positive control. Usually, multiple positive controls should be set up for multiple detection targets, which increases the workload of DNA extraction, and in order to examine DNA quality. , it is necessary to set more reactions of internal standard genes, which further pushes up the detection cost, increases the workload of detection, and causes inconvenience to the detection work
At present, the lack of positive controls (standard samples) for genetically modified screening assays hinders the development of genetically modified assays

Method used

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  • DNA standard sample for transgenic component detection and application of DNA standard sample
  • DNA standard sample for transgenic component detection and application of DNA standard sample
  • DNA standard sample for transgenic component detection and application of DNA standard sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1. Construction of plasmid standard samples

[0069] 1. Determination of ten kinds of foreign gene sequences

[0070] According to a large number of market research data, first determine all the truly commercialized genetically modified lines on the market, and then determine the following seven screening elements as the screening elements that cover the corn DAS40278, soybean DP305423 and soybean CV127 lines: T-E9, T- NOS, P-35S, PAT, T-PinII, P-Rbcs4, T-35S; Finally, we inquire and determine the line-specific sequences of three transgenic lines: corn DAS40278, soybean DP305423 and soybean CV127.

[0071] Through a large number of transgenic information retrieval, sequence comparison, sequence splicing and sequence stability analysis and prediction, the potential sequence complementarity problem between different foreign genes is solved, so that all foreign sequences in the final recombinant plasmid can achieve stable expansion. Finally, the 7 selection elements and ...

Embodiment 2

[0085] Example 2. Preparation of standard samples of standard plasmid molecule pUC57-Exo

[0086] 1. Extraction of plasmid DNA

[0087] The positive strains of the pUC57-Exo plasmid were drawn on a plate to pick out monoclonal colonies, and cultured with shaking at 37°C for 16-18h in 1ml of LB broth containing antibiotics. Aspirate 100μL of culture solution to 100mL of antibiotic-containing LB culture solution for expansion, culture with shaking at 37°C for 16-18h (OD value above 0.8), and collect a total of 100mL bacterial solution. Centrifuge at 6000g for 15 min at 4°C to collect the bacteria. The QIAfilter Plasmid Midi Kits was used for mass extraction and purification of plasmid molecules.

[0088] 2. Plasmid DNA quality evaluation and concentration determination

[0089] Take 1 μL of the extracted plasmid DNA sample and use 1% agarose gel electrophoresis to detect it. If the band is clear and bright, it indicates that the quality of the extracted plasmid DNA is good. See elect...

Embodiment 3

[0123] Example 3. Homogeneity detection of standard samples

[0124] 1. Number of samples

[0125] Fifteen tubes were randomly selected from the candidate standard samples packed into the smallest packaging unit in step 6 of Example 2 for uniformity testing.

[0126] 2. Using fluorescence quantitative PCR method for uniformity detection

[0127] Fluorescent quantitative PCR was performed on 10 exogenous elements. The primers and probes used are shown in Table 4. The working principle of fluorescent quantitative PCR is: Taq DNA polymerase in PCR reaction has 5'→3' exonuclease Active, it can hydrolyze the hybridization probe labeled with fluorescent pigment to release the reporter group and produce fluorescence. The intensity of the fluorescent signal emitted by the reporter group is proportional to the exponentially increasing target DNA fragment. By detecting the fluorescent signal, the PCR products of each reaction stage can be monitored in real time. PCR amplification of 10 kinds ...

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Abstract

The invention discloses a DNA standard sample for transgenic component detection and application of the DNA standard sample. The DNA standard sample comprises DNA fragments shown as SEQ ID NO.1-7, andthe DNA fragments are specific fragments of components T-E9, T-NOS, P-CaMV35S(P-35S), PAT, T-PinII, P-Rbcs4, T-CaMV35S(T-35S). By detection of the seven targets, most of current commercial transgenicvarieties (at least 63) can be covered, and only a transgenic maize DAS40278 transformant and transgenic soybean DP305423 and CV127 transformants without exogenous components fail to be detected. According to detection, uniformity, stability, definite value and the like of the standard sample all meet requirements in detection, and the standard sample can be applied to quality control in daily detection of corresponding exogenous components, verification and evaluation of detection agents, laboratory proficiency testing activity and the like and is available for commercial popularization andapplication.

Description

Technical field [0001] The invention relates to the technical field of genetically modified detection, in particular to a DNA standard sample for detecting genetically modified components and its application. Background technique [0002] In the past 20 years, the global R&D and commercialization of genetically modified organisms has developed rapidly, which has produced huge economic and social benefits. At the same time, the safety of genetically modified organisms has always attracted the attention of all sectors of society. For this reason, more than 60 countries and regions including China, the European Union, the United States, Japan and South Korea have promulgated and implemented laws and regulations on the safety management of genetically modified organisms. Standardize the development and industrial application of genetically modified organisms. [0003] With the rapid development of the genetically modified industry, new conversion events are constantly being introduced...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11C12N15/63
CPCC12N15/63C12Q1/686C12Q1/6895C12Q2600/166C12Q2545/113
Inventor 付伟朱鹏宇王晨光朱水芳
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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