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DNA reference sample containing various endogenous reference gene specific fragments, and application of DNA reference sample

An internal standard gene and standard sample technology, applied in the field of DNA standard samples, can solve the problems of single type of internal standard gene, inconvenient use, high detection cost, etc., to reduce the number of uses, reduce the possibility of missed detection, and have good repeatability Effect

Inactive Publication Date: 2019-11-05
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the plasmid standard contains a single type of internal standard gene. When testing multiple crops at the same time, it is necessary to use multiple plasmids, which is inconvenient to use and high in testing costs.

Method used

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  • DNA reference sample containing various endogenous reference gene specific fragments, and application of DNA reference sample
  • DNA reference sample containing various endogenous reference gene specific fragments, and application of DNA reference sample
  • DNA reference sample containing various endogenous reference gene specific fragments, and application of DNA reference sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, the construction of plasmid standard sample

[0058] 1. Determination of 11 internal standard genes and their specific fragments

[0059] According to a large amount of market research data, first determine the types of transgenic crops for commercial planting, and then determine the following 11 internal standard genes (elements): corn zSSIIb, corn Adh1, soybean Lectin, rape CruA, rape PEP, rice SPS, rice PLD, Generic 18sRNA, Alfalfa ACC, Beet GluA3, Potato UGPase;

[0060] Through a large number of gene information retrieval, sequence alignment, sequence splicing and sequence stability analysis and prediction, we found that the combination of different amplicon sequences can lead to amplification interference between inserts. Therefore, this patent uses real-time fluorescent PCR to verify a large number of standard detection amplicons, evaluates the recombination compatibility of different amplicons through the Ct value obtained by real-time fluorescent...

Embodiment 2

[0075] Embodiment 2, the preparation of the standard sample of standard plasmid molecule pUC57-Ref

[0076] 1. Extraction of plasmid DNA

[0077] Stratify the positive strains of the pUC57-Ref plasmid to pick a single clone colony, culture in 1ml LB culture medium containing antibiotics at 37°C for 16-18h with shaking. Pipette 100 μL of culture solution into 100 mL of LB culture solution containing antibiotics to expand the culture, shake culture at 37°C for 16-18 hours (OD value above 0.8), and collect 100 mL of bacterial solution in total. Centrifuge at 6000 g for 15 min at 4°C to collect the cells. A large number of plasmid molecules were extracted and purified using the QIAfilter Plasmid Midi Kits kit.

[0078] 2. Plasmid DNA quality evaluation and concentration determination

[0079] Take 1 μL of the extracted plasmid DNA sample and detect it by 1% agarose gel electrophoresis. If the bands are clear and bright, it means that the quality of the extracted plasmid DNA is ...

Embodiment 3

[0112] Embodiment 3, the homogeneity detection of standard sample

[0113] 1. Sampling quantity

[0114] 15 tubes were randomly selected from the candidate standard samples subpackaged into the smallest packaging unit in Step 6 of Example 2 for uniformity testing.

[0115] 2. Using fluorescent quantitative PCR method for uniformity detection

[0116] The specific fragments of the 11 internal standard genes were detected by fluorescent quantitative PCR. The primers and probes used are shown in Table 4. The working principle of fluorescent quantitative PCR is: the Taq DNA polymerase in the PCR reaction has a 5'→3' exocut Nuclease activity can hydrolyze the hybridization probe labeled with fluorochrome, thereby releasing the reporter group and generating fluorescence, and the intensity of the fluorescent signal emitted by the reporter group is proportional to the exponentially increasing target DNA fragment. The PCR products of each reaction stage can be monitored in real time ...

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Abstract

The invention discloses a DNA reference sample containing various endogenous reference gene specific fragments, and application of the DNA reference sample. The DNA reference sample contains the specific fragments of eleven types of endogenous reference genes (elements), namely corn zSSIIb, corn Adh1, soybean Lectin, rape CruA, rape PEP, rice SPS, rice PLD, general 18sRNA, alfalfa ACC, sugar beetGluA3 and potato UGPase which are shown as SEQ ID No.1 to 11, and covers seven crops, namely corn, soybeans, rape, potatoes, sugar beet, alfalfa and rice. The using number of positive reference samples in detection is decreased, and the possibility of missing detection is lowered; after detection, it is found that the uniformity, stability, constant value and the like of the reference sample all meet requirements, and the DNA reference sample can be used for quality control over the corresponding endogenous reference genes in daily detection, verification and evaluation for detection reagents,ability verification activities of laboratories and the like, and can be commercially applied and popularized.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a DNA standard sample containing multiple specific fragments of internal standard genes. Background technique [0002] The internal standard gene refers to a conserved DNA sequence that is species-specific, has a constant copy number, and does not show allelic changes. The internal standard gene of a specific species is a genetic marker to distinguish other species, and can be used in many fields such as identification of species and their products, detection of genetically modified ingredients, etc. [0003] The internal standard gene is a necessary detection reference gene in the GMO detection and labeling system. In the detection system, the genomic DNA of the sample to be tested is amplified by PCR technology, and the transferred gene is used as the detection object, and the internal standard gene is used as the control. , the detection results can effectively identif...

Claims

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Application Information

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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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