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Detection method for detecting exogenous gene components of transgenic salmon in animal products by real-time fluorescence quantitative PCR technology

A real-time fluorescence quantitative and exogenous gene technology, applied in the field of bioengineering, can solve problems such as cross-reactions easily, achieve the effects of protecting the right to know and the right to choose, wide application range, and good reproducibility

Inactive Publication Date: 2019-01-18
PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, immunoassays based on antigen-antibody specific binding are prone to cross-reactivity to closely related species

Method used

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  • Detection method for detecting exogenous gene components of transgenic salmon in animal products by real-time fluorescence quantitative PCR technology
  • Detection method for detecting exogenous gene components of transgenic salmon in animal products by real-time fluorescence quantitative PCR technology
  • Detection method for detecting exogenous gene components of transgenic salmon in animal products by real-time fluorescence quantitative PCR technology

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 The design of primer pair and probe

[0039] (1) Design of the first set of primer pairs and probes

[0040] According to the published transgenic salmon exogenous gene (Synthetic construct opAFP-GHc2growth hormone I precursor gene, complete cds) DNA sequence in NCBI (GenBank: AY687640.1), select the appropriate sequence fragments for primer probe design. The length of the target fragment for the exogenous gene components of the transgenic salmon is 139bp.

[0041] The sequence of Synthetic construct opAFP-GHc2growth hormone I precursor gene, completecds DNA sequence (GenBank: AY687640.1) is as follows:

[0042] CTTCGATCC AGATCTTTTCACTTCGATCTCCGATA ATTAATTAATTAATTAATTATTAATTAATTAAGTCTCAGCCACTGCAGGTCGTAAAA ATGGGACAAGTGTTTCT GCTGATGCCAGTCT TACTGGTCAGTTGTTTCCTGA GTCA AGGGGCAGCG (SEQ ID NO: 1).

[0043] The sequences of fluorescent PCR primer pairs and probes are as follows:

[0044] GMS-139bp-F: 5'-AGATCTTTTCACTTCGATCTCCGATA-3' (SEQ ID NO: 2);

[0...

Embodiment 2

[0067] The preparation of embodiment 2 test samples

[0068] After the animal samples were shredded and dried, they were ground into powder using a freeze grinder (SPEX 6850). Plant samples and samples for practical validation were directly ground into powder using a cryo-mill (SPEX 6850).

Embodiment 3

[0069] The extraction of embodiment 3 DNA

[0070] DNA from animal samples was extracted using the Animal Genome Extraction Kit (Tiangen Biochemical Technology Co., Ltd.; catalog number: DP323), and DNA from plant samples was extracted using the Plant Genome Extraction Kit (Tiangen Biochemical Technology Co., Ltd.; catalog number: DP305). Genetic Plant Feed Genome Extraction Kit (Tiangen Biochemical Technology Co., Ltd.; catalog number: DP323) was used to extract DNA from mixed samples and actual verification samples. For the extraction method, refer to the kit operation manual for details. The DNA solution was stored at -20°C for later use.

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Abstract

The invention discloses a detection method for detecting exogenous gene components of transgenic salmon in animal products by real-time fluorescence quantitative PCR technology. The method comprises the following steps: firstly, taking DNA of a sample to be detected as a template, performing fluorescent quantitative PCR amplification to obtain PCR amplification products; and secondly, detecting the exogenous gene components of the transgenic salmon by using a real-time fluorescent quantitative PCR technology. 2, detecting fluorescence signals of that amplification product; 3, judging whether that sample contain the foreign gene component of the transgenic salmon according to the Ct value of the detection result; Among them, the reaction system used for PCR amplification contains specific primer pairs and specific probes for amplifying foreign gene components of transgenic salmon. The specific primer pair and probe of the foreign gene component of the transgenic salmon of the inventionare not only good in specificity but also high in sensitivity, which provides a quantitative detection method for quickly and accurately detecting whether the foreign gene component of the transgenicsalmon is contained in the animal products, and has a good application prospect.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for detecting exogenous gene components of transgenic salmon in animal products by using real-time fluorescent quantitative PCR technology. Background technique [0002] On November 19, 2015, after confirming its food safety and environmental safety, the U.S. Food and Drug Administration (FDA) approved the marketing of AquaBounty's genetically modified salmon strain "AquAdvantage", making it the first to be approved. Batches of genetically modified animals for food. The main characteristic of this kind of salmon is that it grows rapidly, and compared with conventional salmon (Atlantic salmon, Salmo salar), it saves one and a half years of breeding time, thereby greatly reducing the cost. This kind of salmon adopts a pure land-based cultivation method. The eggs are cultivated in Canada while the farming and slaughter are carried out in Panama. The fish is shippe...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q2531/113C12Q2563/107C12Q2545/113
Inventor 蔡一村王强林颖峥吕蓉潘良文
Owner PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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