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Method for identifying squids or highly processed product varieties thereof by virtue of fluorescent quantitative PCR

A fluorescent quantitative and product variety technology, applied in the field of fluorescent quantitative PCR detection, can solve the problems of low cost, messy sequencing results, affecting polymorphism of enzyme fragments, etc., and achieve the effect of short detection time, easy operation and good specificity

Inactive Publication Date: 2016-04-27
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is low in cost and easy to operate, but it also has many disadvantages: 1), due to the existence of intraspecies variation and the degeneracy characteristics of different codons, different individuals of the same species may have different restriction fragment digestion patterns; 2), except In addition to fresh squid and frozen squid, deep-processed squid products (such as squid shreds, squid cakes, canned squid, etc.) are usually treated at high temperature (>115°C) for a long time (>15min) during processing, and DNA molecules are often degraded to 100- A small fragment of 200bp, which may seriously affect the judgment of the polymorphism of the restriction fragment
This method is very efficient in the discovery of new species and the analysis of species phylogeny, but its disadvantage is that multiple components in mixed samples are usually amplified at the same time, which will cause confusion in the sequencing results, so it is not suitable for the identification of mixed samples

Method used

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  • Method for identifying squids or highly processed product varieties thereof by virtue of fluorescent quantitative PCR
  • Method for identifying squids or highly processed product varieties thereof by virtue of fluorescent quantitative PCR
  • Method for identifying squids or highly processed product varieties thereof by virtue of fluorescent quantitative PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Embodiment 1, adopt above-mentioned fluorescence quantitative PCR detection method to the squid frozen product (such as frozen squid ring, frozen squid carcass) species identification of buying randomly on the market, comprise carrying out the following steps:

[0090] ①. Genomic DNA extraction from the squid sample to be tested; Axygen Genomic DNA Mini-Extraction Kit was used to extract sample DNA and store at -20°C.

[0091] ②. Design of primers and probes for specific identification of common squid;

[0092] The 4 common squid are: North Pacific squid (Ommastrephesbartramii), Peruvian squid (Dosidicusgigas), Argentine squid (Illexargentinus) and Japanese sea squid (Todarodespacificus);

[0093] Specifically as described in Table 1.

[0094] ③. Fluorescence quantitative PCR amplification system setting;

[0095] Using genomic DNA as an amplification template, the above-mentioned specific primers and probes are used for real-time fluorescent PCR detection.

[0096] ...

Embodiment 2

[0112] Embodiment 2, adopt above-mentioned fluorescent quantitative PCR detection method to carry out kind identification to the canned squid food that buys randomly on the market:

[0113] The canned food labeled as canned squid or canned squid was randomly purchased from the market, and the sample DNA was extracted with TAKARA Meat DNA Extraction Reagent (Cat#9178) and stored at -20°C. The species were identified by fluorescent quantitative PCR. If there is no amplification curve for the 12S rDNA detection gene of the Squididae or C t If the value is ≥ 34, the determination result is negative, and it can be determined that the canned fish sample does not contain fish components of the family Scutellaria; C t If the value is less than 34, it can be determined that the sample result is positive, that is to say, it is determined that the sample contains fish components of the family Scutellaria. If the specificity system tests of the four common squids have no amplification c...

Embodiment 3

[0119] Embodiment 3, adopt above-mentioned fluorescent quantitative PCR detection method to carry out species identification to the deep-processed squid food that buys randomly on the market:

[0120] Randomly purchased from the market marked as squid shreds, squid slices and products containing squid in the product ingredients, TAKARA meat product DNA extraction reagent (Cat#9178) was used to extract sample DNA, and stored at -20 °C. The species were identified by fluorescent quantitative PCR. If there is no amplification curve for the 12S rDNA detection gene of the Squididae or C t If the value is ≥ 34, the judgment result is negative, and it can be judged that the deep-processed product does not contain fish components of the family Scutellaria; C t If the value is less than 34, it can be determined that the sample result is positive, that is to say, it is determined that the sample contains fish components of the family Scutellaria. If there is no amplification curve in ...

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Abstract

The invention discloses a method for identifying squids or highly processed product varieties thereof by virtue of fluorescent quantitative PCR. The method comprises the following steps: (1) extracting genome DNA from a to-be-detected squid sample; (2) designing specific identification primer and probe for common squids, wherein the four common squids include Ommastrephes bartramii, dosidicus gigas, illex argentinus and todarodes pacificus; (3) setting a fluorescent quantitative PCR amplification system; and conducting real-time fluorescent PCR detection by virtue of the specific primer and probe by taking the genome DNA as an amplification template; and (4) judging in accordance with the result of the real-time fluorescent PCR detection. By virtue of the method disclosed by the invention, the squids and the highly processed product varieties thereof can be rapidly identified.

Description

technical field [0001] The invention relates to a fluorescent quantitative PCR detection method suitable for squid and its deep-processed products. Background technique [0002] Squid, also known as Jugong, soft fish or squid, is a common name for a variety of 10 brachiopod molluscs in Cephalopoda and Teuthida. Its body is elongated and gun-shaped. The olfactory process is composed of two protrusions, the outer orbit is covered with transparent cornea, and the ring mouth is appended to 10 wrists. Strong water jet propulsion ability, suitable for fast swimming. Squid are widely distributed in all oceans of the world, and the known water layer is 0-3000m. Common squids on the market are generally the Ommastrephidae species of the Oegopsida suborder and the Loliginidae species of the Myopsida suborder. In the domestic market of China, the most common squid are Peruvian squid (Dosidicus gigas), Argentine squid (Illexargentinus), Japanese sea squid (Todarodes pacificus) and N...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2563/107C12Q2545/114C12Q2531/113
Inventor 冯俊丽叶剑戴志远金仁耀沈清
Owner ZHEJIANG GONGSHANG UNIVERSITY
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