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Dual fluorescent quantitative PCR method for species identification of salmons and highly processed products of salmons

A dual fluorescence and product variety technology, applied in the field of dual fluorescence quantitative PCR detection, can solve the problems of low cost, messy sequencing results, and inapplicability of mixed sample identification, and achieve the effect of shortening the detection time and reducing the cost of reagents

Inactive Publication Date: 2015-11-11
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 of different codons, different individuals of the same species may have different restriction fragment digestion patterns; 2) In addition to raw fish Outside of slices, deep salmon products (such as canned salmon) are usually processed at high temperature (>115°C) for a long time (>15min) during processing, and DNA molecules are often degraded into small fragments of 100-200bp, which may seriously affect enzyme-digested fragments polymorphic judgment
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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  • Dual fluorescent quantitative PCR method for species identification of salmons and highly processed products of salmons
  • Dual fluorescent quantitative PCR method for species identification of salmons and highly processed products of salmons
  • Dual fluorescent quantitative PCR method for species identification of salmons and highly processed products of salmons

Examples

Experimental program
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Effect test

Embodiment 1

[0090] Embodiment 1, adopt above-mentioned double fluorescence quantitative PCR detection method to carry out species identification to the salmon sashimi that buys randomly on the market:

[0091] 1) Design of primers and probes for specific identification of Salmonidae and 4 common salmon species;

[0092] Specifically as described in Table 1.

[0093] 2), the extraction of genomic DNA in the salmon sample to be tested;

[0094] The sashimi marked as salmon was randomly purchased from the market, and the sample DNA was extracted with the Axygen Genomic DNA Mini-extraction Kit, and stored at -20°C.

[0095] 3) Fluorescence quantitative PCR amplification system setting:

[0096] The species were identified by double fluorescent quantitative PCR.

[0097] The dual fluorescent quantitative PCR system is: AceQ is contained in a total volume of 20 μL TM qPCRProbeMasterMix 10 μL, 0.4 μL each of salmon-specific upstream and downstream primers (5 μM), 0.2 μL of salmon-specific pr...

Embodiment 2

[0111] Embodiment 2, using the above-mentioned double fluorescent quantitative PCR detection method to carry out the species identification of the salmon and fish floss commodities purchased randomly on the market:

[0112] Randomly purchased goods marked as salmon fish floss from the market were used to extract sample DNA using Tiangen Deep Processed Food DNA Extraction Kit (DP326), and stored at -20°C. The species were identified by double fluorescent quantitative PCR. If the 16S rDNA detection gene of Salmonidae has no S-type amplification curve and / or the Ct value is greater than 30, the judgment result is negative, and it can be determined that the fish floss sample does not contain salmonid fish components; if the Ct value is less than 30, it can be judged as negative. If the result of the sample is determined to be positive, it is determined that the sample contains components of salmonids. If the CR detection genes of the 4 common salmon species have no S-curve genera...

Embodiment 3

[0119] Embodiment 3, using the above-mentioned double fluorescent quantitative PCR detection method to carry out the species identification of the canned salmon fish goods purchased randomly on the market:

[0120] Randomly buy canned salmon products from the market, use Tiangen Deep Processed Food DNA Extraction Kit (DP326) to extract DNA from samples, and store them at -20°C. The species were identified by double fluorescent quantitative PCR. If the 16S rDNA detection gene of Salmonidae has no S-type amplification curve and / or the Ct value is greater than 30, the judgment result is negative, and it can be determined that the canned fish sample does not contain salmonid fish components; if the Ct value is less than 30, it can be determined If the result of the sample is determined to be positive, it is determined that the sample contains components of salmonids. If the CR detection genes of the 4 common salmon species have no S-curve generation and / or the Ct value is greater...

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Abstract

The invention provides a dual fluorescent quantitative PCR method for species identification of salmons and highly processed products of the salmons, and discloses a method for detecting the species of the salmons and the highly processed products of the salmons through dual fluorescent quantitative PCR. The method includes the following steps that firstly, specific identification primers and probes of salmonidae and four kinds of common salmons are designed, wherein the four kinds of common salmons include atlantic salmons, rainbow trouts, dog salmons and sockeye salmons; secondly, genome DNA in a salmon sample to be detected is extracted; thirdly, a dual fluorescent quantitative PCR amplification system is set; fourthly, judgment is made according to real-time fluorescent PCR detection results. The effective method is provided for quick detection of species identification of the salmons and the highly processed products of the salmons, and a technological guarantee is provided for aquatic product and food import and export.

Description

technical field [0001] The invention relates to a double fluorescent quantitative PCR detection method suitable for identification of salmon and its deep-processed products. Background technique [0002] Salmon is a general term for salmonid fish or salmon trout, and is known as "the supreme fish" and "treasure in water". Salmon is mainly produced in the border waters of the Arctic Ocean, the Atlantic Ocean, and the Pacific Ocean, and belongs to migratory fish in cold waters. Norway, which borders the Arctic Ocean, is the world's largest salmon farming and fishing country, accounting for half of the world's total production. Norwegian salmon is known as the "Emperor of the Ice Sea" by the international food industry for its best quality and best taste. At present, the domestically recognized economic species are roughly divided into 3 subfamilies, 7 genera, and about 36 species. As a commodity name, salmonids can be called salmon as long as the muscles are firm and elasti...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 冯俊丽戴志远吴志刚谢潇刘莎莎
Owner ZHEJIANG GONGSHANG UNIVERSITY
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