Fluorescent quantitative PCR method for detecting fish parvalbumin and primer pair

A technology of parvalbumin and fluorescence quantification, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. problems, to achieve the effect of low detection cost, simple result judgment, and reliable detection results

Active Publication Date: 2016-02-24
ZHEJIANG ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The current detection methods for fish parvalbumin mainly include serological testing and protein characteristic methods, among which, serological methods (RAST, CAP, etc.) The allergen solid-phase carrier has different binding ability to IgE, so it is more difficult to commercialize food allergen detection; and

Method used

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  • Fluorescent quantitative PCR method for detecting fish parvalbumin and primer pair
  • Fluorescent quantitative PCR method for detecting fish parvalbumin and primer pair
  • Fluorescent quantitative PCR method for detecting fish parvalbumin and primer pair

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Embodiment 1

[0023] Embodiment 1: detect the real-time fluorescent quantitative PCR method of perch parvalbumin, specifically comprise the following steps:

[0024] Step 1, designing primers according to the conserved sequence in the genome DNA sequence encoding fish parvalbumin

[0025] Through the analysis of amino acid sequence and coding gene sequence, because fish parvalbumin has good conservation, primers were designed according to the specificity of the protein coding gene. First find the parvalbumin-encoding genes of different fish in GenBank, and find out the conserved segments after comparison analysis, and then compare the selected conserved segments with the DNA sequences of other animal species in GenBank by Blast software, Find the non-homologous region of DNA with other species, and use the primer design software PrimerPremier5.0 to design specific primers for parvalbumin therefrom. The primer sequence is as follows (primers are provided by Dalian Bao Biological Engineering ...

Embodiment 2

[0038] Embodiment 2: Utilize the detection method of embodiment 1, to chicken, beef, donkey meat, mutton, duck, pork 6 kinds of non-fish meat products, and small yellow croaker, large yellow croaker, salmon, sea bream, red fish fillet, cod 6 kinds of fish products were tested for parvalbumin gene, the results are as follows image 3 , 4As shown (1-12 in the figure respectively represent: 1. Chicken; 2. Beef; 3. Donkey meat; 4. Mutton; 5. Duck; 6. Pork; 7. Small yellow croaker; 8. Large yellow croaker; 9. Salmon ; 10. sea bream; 11. red fish fillet; 12. cod), 6 kinds of non-fish meat products were all negative; the test results of the remaining 6 kinds of fish meat products were all positive.

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Abstract

The invention relates to a fluorescent quantitative PCR method for detecting fish parvalbumin and a primer pair and belongs to the technical field of food safety detection. The PCR method comprises the following steps: I, designing a specific amplification primer pair; II, extracting DNA of a sample; III, judging whether the sample contains a parvalbumin gene or not through an amplification curve and a dissolution curve; the invention further relates to the primer pair. To be specific, the primer pair is as follows: base sequences of the fish parvalbumin are shown as SEQ ID No.1 and SEQ ID No.2. Compared with the prior art, the fluorescent quantitative PCR method and the primer pair have the advantages as follows: by using bioinformatics and comparative genomics, the shared gene sequences of fish parvalbumin are selected, the specific amplification primer pair is designed according to the sequences, the primer pair provided by the invention can be used for carrying out fluorescent quantitative PCR detection on samples to detected and can rapidly, sensitively and specifically detect the allergen ingredient namely parvalbumin in different kinds of fishes and fish products in food.

Description

technical field [0001] The invention relates to the technical field of food safety detection, in particular to a fluorescent quantitative PCR method and a primer pair for detecting fish parvalbumin. Background technique [0002] In 1969, Aas and Elsayed identified the main allergen of Pacific cod (Gaduscallarias) for the first time and named it Gadc1, which was one of the first identified food allergens in the world. Gadc1 is composed of 113 amino acids and 1 glucose molecule, with a molecular weight of about 12kD. It belongs to parvalbumin and is a calcium-binding protein. This parvalbumin exists in large quantities in the white muscles of lower vertebrates. Parvalbumin can be divided into two different types according to the difference in amino acid sequence: α-type parvalbumin pI≥5.0, containing a small amount of acidic parvalbumin, and β-type parvalbumin pI≤4.5, with higher acidic parvalbumin content. Most of the parvalbumin in fish belong to the β type. [0003] Fish ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 李可张晓峰张明哲朱晓雨方莹
Owner ZHEJIANG ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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