Real-time polymerase chain reaction (PCR) detection primer, kit and detection method for detecting puffer fish ingredients

A detection method, the technology of puffer fish, applied in the field of molecular biology, can solve the problems of limited versatility, achieve the effects of protecting food safety, wide application prospects, and improving detection efficiency and sensitivity

Inactive Publication Date: 2013-05-22
曹际娟 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Real-time polymerase chain reaction (PCR) detection primer, kit and detection method for detecting puffer fish ingredients
  • Real-time polymerase chain reaction (PCR) detection primer, kit and detection method for detecting puffer fish ingredients
  • Real-time polymerase chain reaction (PCR) detection primer, kit and detection method for detecting puffer fish ingredients

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 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. The DNA extracted from puffer fish was used as positive control, the DNA extracted from samples known not to contain puffer fish components was used as negative control, and sterilized water was used as blank control. Two parallel reaction systems were set up for each group.

[0038] 1. DNA extraction

[0039] For the following DNA extraction methods, all reagents and solutions were prepared in conventional ways or obtained from commercial sources.

[0040] 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 ...

Embodiment 2

[0050] Embodiment 2 specificity test

[0051] ①Real-time PCR specific amplification of cytochrome b gene fragment of puffer fish:

[0052] Take the DNA of 42 samples listed in Table 2, and use Puffer F / Puffer R primers and Puffer P probes for real-time PCR detection. The real-time PCR amplification pattern for specific detection of puffer fish is as follows: figure 1 shown.

[0053] 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.

[0054] Table 2 Sample name and source

...

Embodiment 3

[0059] The sensitivity of embodiment 3 detection method

[0060] The processing of fish products will cause DNA breakage and loss, which will also affect the sensitivity of detection. In order to study the influence of the processing process on the detection sensitivity, this experiment took monkfish as the substrate, added puffer fish, and did the addition test of simulated processed products.

[0061] Puffer fish and monkfish samples were taken respectively, minced in a meat grinder, baked overnight at 80°C, and ground into powder. Puffer fish meal and monkfish meal samples were mixed according to the mass ratio of puffer fish at 100%, 10%, 5%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, to obtain a series of mixed samples of fish meal with different content According to the method described in Example 1, real-time PCR detection sensitivity analysis was carried out using Puffer F / Puffer R primers and Puffer P probes. The result is as figure 2 shown. From the process of baking sample...

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Abstract

The invention discloses a real-time polymerase chain reaction (PCR) detection primer, a kit and a detection method for detecting puffer fish ingredients. The puffer fish gene detection primer and probes with the sequences of SEQ ID NO. 1-3 are designed according to the specific gene sequence of puffer fish, and the real-time fluorescent PCR method is adopted to qualitatively detect the puffer fish ingredients of food. The developed real-time fluorescent PCR qualitative detection method for detecting the puffer fish ingredients in food has importance significance in improving the detection efficiency and sensitivity of the puffer fish ingredients in food, lowering the detection cost, detecting related fake products on the market, monitoring food safety and improving a detection technology, and has wide development prospect.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to real-time PCR detection primers, a kit and a detection method for the detection of puffer 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, til...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 曹际娟李晶泉郑秋月徐君怡
Owner 曹际娟
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