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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 can quickly, simply, specifically and sensitively detect shark fins.

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

Embodiment 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 the partial sequence of shark mitochondrial 16S rDNA obtained by PCR cloning and sequencing.

[0030] According to the difference of mitochondrial 16S rDNA sequences in different sharks and teleosts, the upstream and downstream primers were designed to amplify star sharks. According to the instructions of Wizard Gel Extraction Kit (Promega, USA), the star shark PCR product was purified and recovered. According to the instructions of the TaKaRa pMD19-TVector kit (TaKaRa, Japan), the purified product was connected to pMD19-T Vector, the connection system was 10 μL, and the reaction components were: pMD19-T Vector 1 μL, PCR product 2 μL, ddH 2 O 2 μL, Solution I 5 μL, set positive and negative controls at the same time. Place the connection system at room temperature (22-37° C.) for 30 minutes and place it on...

Embodiment 2

[0036] In this embodiment, the extraction quality of the total DNA of a sample is tested by using a general primer pair and a probe for sharks and teleosts.

[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 technology for real-time fluorescent quantitative detection of single-tube PCR products. In an ordinary PCR amplification system, a dual fluorescent-labeled probe that is specifically complementary to the target gene sequence is added to utilize the fluorescent signal. Accumulate and monitor the entire PCR process in real time, and finally perform quantitative analysis on the unknown template through the standard curve. Quantitative steps: ① determine the CT value (C represents the cycle number (Cycle), T represents the fluorescence threshold value (Threshold), that is, the number of cycles experienced when the fluorescence signal in each rea...

Embodiment 3

[0066] In this example, the specificity and sensitivity of shark primer pairs and probes were verified through the following tests.

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

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

[0069] The primer sequences are SEQ ID No.1 and SEQ ID No.2, the probe sequence is SEQ ID No.3, a fluorescent 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 invention, compared with other p...

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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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