Primer, probe, kit and method for real-time fluorescence PCR identification of shark's fin

A real-time fluorescence and kit technology, applied in the fields of biochemical equipment and methods, fluorescence/phosphorescence, and microbial determination/inspection, to achieve the effects of simple operation, avoidance of cross-contamination, and reliable results

Inactive Publication Date: 2011-02-16
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are few reports at home and abroad that c

Method used

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  • Primer, probe, kit and method for real-time fluorescence PCR identification of shark's fin
  • Primer, probe, kit and method for real-time fluorescence PCR identification of shark's fin
  • Primer, probe, kit and method for real-time fluorescence PCR identification of shark's fin

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0027] Example 1

[0028] The inventors of the present invention cloned and sequenced the partial sequence of shark mitochondrial 16S rDNA by PCR for the first time.

[0029] This example is a partial sequence of shark mitochondrial 16S rDNA obtained by PCR cloning and sequencing.

[0030] According to the characteristics of mitochondrial 16S rDNA sequence in different sharks and bony fishes, the upstream and downstream primers were designed to amplify the star shark. Purify and recover the PCR product of Xing Shark according to the operating instructions of Wizard Gel Extraction Kit (Promega, USA). Connect the purified product to pMD19-T Vector according to the instructions of the TaKaRa pMD19-TVector kit (TaKaRa, Japan). The connection system is 10μL. The reaction components are: pMD19-T Vector 1μL, PCR product 2μL, ddH 2 O 2μL, Solution I 5μL, and set positive and negative controls at the same time. The connection system was placed at room temperature (22-37°C) for 30 minutes, a...

Example Embodiment

[0035] Example 2

[0036] In this example, the extraction quality of the total DNA of the sample was tested by using universal primer pairs and probes for sharks and bony fishes.

[0037] By detecting the mitochondrial 16S rDNA sequence, the extraction quality of the total DNA of the sample can be tested. Taqman fluorescent probe method: TaqMan technology is a real-time fluorescent quantitative detection technology for single-tube PCR products. In the ordinary PCR amplification system, a double fluorescent probe that is specifically complementary to the target gene sequence is added to use the fluorescent signal Accumulate real-time monitoring of the entire PCR process, and finally quantitatively analyze the unknown template through the standard curve. Quantitative steps: ①Determine the CT value (C represents the cycle number (Cycle), T represents the fluorescence threshold (Threshold), that is, the number of cycles experienced when the fluorescence signal in each reaction tube re...

Example Embodiment

[0065] Example 3

[0066] In this example, the specificity and sensitivity of the primer pairs and probes of sharks were verified by the following experiments.

[0067] By detecting the mitochondrial 16S rDNA sequence, the specificity and detection sensitivity of the shark primer and probe combination can be determined. The reaction system is: Fast Start Universal PCR Master Mix 12.5μL; probe (10μM) 0.5μL; upstream and downstream primers (10μM) each 0.5μL; template DNA 5μL; add ddH 2 O to a total volume of 25μL. The reaction program is 95°C for 10 minutes; 95°C for 15 seconds; 60°C for 1 minute, 40 cycles.

[0068] The primers and probe sequences used to detect shark fin are:

[0069] The primer sequence is SEQ ID No. 1 and SEQ ID No. 2, the probe sequence is SEQ ID No. 3, a fluorescence quenching group TAMRA is connected to the 3'end, and a fluorescent reporter group HEX is connected to the 5'end.

[0070] Using the combination of the primer pair and the probe of the present inventio...

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Abstract

The invention relates to an oligonucleotide primer and a probe for shark's fin reality identification. The invention also relates to a real-time fluorescence PCR detection method for shark's fin reality identification. The specific oligonucleotide primer and the probe are used in the method. The invention also relates to a PCR detection kit for shark's fin reality identification. The kit comprises the specific oligonucleotide primer and the probe. The invention also relates to application of the specific oligonucleotide primer and the probe in identifying shark's fin reality. The PCR detection method can simply, quickly, specifically and sensitively identify the reality of the shark's fin.

Description

technical field [0001] The invention belongs to the field of biotechnology, specifically, the present invention relates to oligonucleotide primers and probes for identifying the authenticity of shark fins, a kit comprising the oligonucleotide primers and probes of the present invention, which is used to identify shark fins A real-time fluorescent PCR detection method for authenticity, and the application of the specific oligonucleotide primer and probe or kit in identifying the authenticity of shark fins. Background technique [0002] Shark's fin is a traditional high-end food in China, listed as one of the eight treasures, known as "balone, ginseng, fin, and belly", and its consumption is increasing year by year. There have been records of eating shark fin in China since the Song Dynasty at the latest. "Song Huiyao" has a record about Fujian's import of shark fin from overseas. By the Ming Dynasty, it was widely eaten, and it was recorded in books such as "Qianque Leishu"...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 陈颖韩建勋邓婷婷黄文胜
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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