HRM primer and method for identification of puffer fishes and other fish products

A technology for fish meat products and puffer fish, which is applied in biochemical equipment and methods, microbiological measurement/inspection, DNA/RNA fragments, etc., can solve problems such as difficulty in obtaining COI sequences, and achieve high specificity and strong identification capabilities

Inactive Publication Date: 2018-12-07
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the DNA of commercially available fish meat products is highly degraded, and it is difficult to obtain the full-length COI sequence

Method used

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  • HRM primer and method for identification of puffer fishes and other fish products
  • HRM primer and method for identification of puffer fishes and other fish products
  • HRM primer and method for identification of puffer fishes and other fish products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 3 kinds of DNA barcode identification of pufferfish of embodiment 1

[0038] 1.1 Experimental materials, reagents and instruments

[0039] The experimental materials were 4 common puffer species: 18 yellow-fin puffers, 18 obscurate puffers, 18 chrysanthemum-yellow puffers, and 19 red-finned puffers. At the same time, saury (Cololabis saira) was used as the outgroup.

[0040] The main reagents are: sterilized water, marine animal tissue genomic DNA extraction kit (Tiangen Biochemical Technology, DP324), primers (Shanghai Sangong), PCR kit (Japan TAKARA, Ex Taq), agarose (Shanghai Sangong, Low electroosmotic agarose B), nucleic acid dye (Beijing Zhuangmeng, GoldView), DNAmarker (Japan TAKARA, DL2,000).

[0041] The main instruments are: sterilizing pot (Shanghai Lidetai, LT-CPS50C), oven (Shanghai Boxun, GZX-9023MBE), electronic balance (Changzhou Ohaus Instruments, AR224CN), homogenizer (Hangzhou Aosheng Instruments , Bioprep-24), constant temperature water bath (Hang...

Embodiment 2

[0106] Example 2 Rapid identification method of pufferfish and other fish products based on COI gene

[0107] Combined with the optimal DNA barcode COI sequence obtained in Example 1 and the four identified puffer species that have been obtained, this example provides a rapid identification method for pufferfish and other fish products based on the COI gene in combination with HRM technology

[0108] 2.1 Experimental materials, reagents and instruments

[0109] Materials: 9 commercially available puffer fish foods, 5 commercially available non-puffer fish foods, and 10 fish foods without clearly marked sources. See Table 5 for specific types:

[0110] Table 5 Sample table of fish and meat products for sale

[0111]

[0112]

[0113] Reagents: NaCl, Tris Base, Na 2 -EDTA, CTAB, Tris saturated phenol (pH=8.0), isoamyl alcohol, ethanol, isopropanol, HRM analysis kit (Tiangen Biochemical Technology, FP210).

[0114] Instruments: thermostatic metal bath (Hangzhou Aosheng ...

Embodiment 3

[0143] Example 3 Rapid Identification Result and Analysis of Several Commercially Available Fish and Meat Products

[0144] Based on Examples 1 and 2, we used two pairs of primers to perform HRM analysis on 9 common "puffer fish meat" products sold in the market to detect whether the label on the food package is true. The standard samples are composed of the above-mentioned yellowfin fugu, obscurus fugu, chrysanthemum yellow fugu and red fin fugu, which are represented by dotted lines, solid lines, long and short lines and dashed lines, respectively, and the gray dotted lines represent the "river Fish meat" products. Amplification curve such as Figure 9 As shown, except for individual outliers, comparing the two amplification curves, all samples have obvious amplification curves, and the samples are preliminarily determined to be Pufferfish.

[0145] High resolution melting curves such as Figure 9 As shown, the curve of primer COIHRM168 redfin puffer is looser than that o...

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Abstract

The invention provides an application of an HRM primer for identification of puffer fishes and other fish products, wherein the HRM primer has the following sequences: a forward primer COIHRM168 is 5'-CCCCTTATAATCGGAGCCCC-3' and a reverse primer COIHRM255 is 5'-GCAGAAGGAGGAAGGATGGG-3'; or forward primer COIHRM175 is 5'-TAATCGGAGCCCCAGACATG-3' and a reverse primer COIHRM262 is 5'-GATGCGAGCAGAAGGAGGAA-3'.

Description

technical field [0001] The invention relates to a method for identifying puffer fish, in particular to an HRM primer and a method for identifying puffer fish and other fish products. Background technique [0002] Food safety is related to the national economy and people's livelihood, and food species identification is an important link in the food safety chain. Nature is rich in species, and certain species can pose a threat to the health of specific groups of people, such as various types of poisonous animals and plants, allergens and pathogenic bacteria. Foods with unknown sources of raw materials or unknown ingredients will pose a huge risk to food safety. Pufferfish (English: Pufferfish), commonly known as lungfish, air drum fish or air fish, etc., is a member of the family Diodontidae, Triodontidae and Tetraodontidae in the order Tetraodontiformes. ) and a general term for fish belonging to the family Ostraciontidae. Pufferfish are often mistakenly written as "puffer...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2527/107C12Q2531/113C12Q2563/107
Inventor 丁艳菲朱诚王熠孙骏威王飞娟蒋晗江琼
Owner CHINA JILIANG UNIV
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