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Fish composition detection real-time PCR (polymerase chain reaction) detection primer, kit and detection method

A technology for component detection and detection method, applied in the field of molecular biology, can solve problems such as versatility limitations, and achieve the effects of protecting food safety, wide application prospects, and improving detection efficiency and sensitivity

Inactive Publication Date: 2014-05-21
曹际娟 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method can only analyze one species with a pair of specific primers, which is greatly limited in versatility

Method used

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  • Fish composition detection real-time PCR (polymerase chain reaction) detection primer, kit and detection method
  • Fish composition detection real-time PCR (polymerase chain reaction) detection primer, kit and detection method
  • Fish composition detection real-time PCR (polymerase chain reaction) detection primer, kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] In the testing process, in order to ensure the credibility of the experimental results, a positive control, a negative control, and a blank control group were respectively set up. DNA extracted from fish was used as a positive control, DNA extracted from a sample known not to contain fish components was used as a negative control, and sterilized water was used as a blank control. Two parallel reaction systems were set up for each group.

[0037] 1. DNA extraction

[0038] For the following DNA extraction methods, all reagents and solutions were prepared in conventional ways or obtained from commercial sources.

[0039] Sample DNA was extracted using the improved high-salt method. Grind the sample with liquid nitrogen, take 150 mg of the ground sample into a 1.5 mL centrifuge tube, add 400 μL of TE (containing 10 mmol / L Tris-HCl and 1 mmol / L EDTA), 40 μL of 10% SDS and 8 μL of 20 mg / mL protease K, vortex for 30s, and mix thoroughly; place the centrifuge tube in a const...

Embodiment 2

[0049] Embodiment 2 specificity test

[0050] ①Real-time PCR-specific amplification of fish 12s DNA fragments:

[0051] Take the sample DNA listed in Table 2, and use Fish F / Fish R primers and Fish P probes to perform real-time PCR detection according to the method in Example 1. Real-time PCR amplification profiles for fish species-specific detection such as figure 1 shown.

[0052] Samples and sources described in the embodiments of the present invention: 3 kinds of monkfish (Lophius litulon, Lophiomus setigerus, Lophius americanus), 9 kinds of puffer fish (Lagocephalus inermis, Lagocephalus lagocephalus, Takifugufasciatus, Takifugu xantopterus, Takifugu oblongus, Takifugu rubripes, Gastro gloveri, Gastrophysus lunaris, Gastrophysus spadiceus, and other 24 fish samples, 1 octopus (Octopus), 1 cod crab (Chionoecetes bairdi), 2 shrimps, 2 shellfish samples, a total of 42 samples. Specific samples See Table 2 for names and sources.

[0053] Table 2 Sample name and source

...

Embodiment 3

[0057] Application of embodiment 3 method

[0058] The actual test samples are selected, and the source of the samples is the inspection import and export samples, the samples collected from the Agricultural Science Institute, the food purchased from various supermarkets, markets, food stalls, etc. in Dalian City, etc., are tested for fish source components by the method of the present invention. The method described in Example 1 of the present invention is used for detection, and the amplification curve is determined according to the result determination standard described in Example 1; the sample names and results are shown in Table 3.

[0059] Table 3 actual sample test results

[0060]

[0061] As can be seen from the above detection results, a total of 168 actual samples were detected using the method of the present invention. Detect fish fillets, etc. (53), canned food, etc. (60), and snacks (55), of which 53 fish fillets, etc. were detected to contain fish component...

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Abstract

The invention discloses a real-time florescent PCR (polymerase chain reaction) detection primer of fish composition in food, a kit and a detection method. The method comprises the following steps of: designing a primer for detection of fish genes and a probe SEQ ID (sequence identification) NO.1-3 aiming at the specific gene sequence of fish; and detecting fish composition in food quantitatively by adopting a real-time florescent PCR method. The real-time florescent PCR method quantitative detection method of fish composition in researched food has great significance for improving the detection efficiency and flexibility of fish composition in food, lowering the detection cost, detecting relevant false products in the market, monitoring food safety, and improving detection technique, and has wide development prospect.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a real-time PCR detection primer, a kit and a detection method for detecting fish components in food. Background technique [0002] Aquatic products are an important part of the world economy and trade, and also an important source of food protein. There are many kinds of edible aquatic products, and there are 25 major species in the main species of international trade. There are more than 1,700 kinds of edible aquatic products listed by the US Food and Drug Administration alone. With the development of international trade of aquatic products and the improvement of processing level, the relationship between supply and demand of certain types of products has changed, and some merchants have sold low-value aquatic products as high-value aquatic products to obtain improper economic benefits, such as counterfeiting farmed trout Atlantic salmon, tilapia, mahi ma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 曹际娟李晶泉徐君怡徐杨
Owner 曹际娟
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