Primer, probe, kit and method for identifying authenticity of cervus elaphus linnaeus

A red deer antler and kit technology, applied in biochemical equipment and methods, microbe determination/testing, DNA/RNA fragments, etc., to achieve the effects of avoiding cross-contamination, simple operation, accurate and sensitive 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

However, up to now, the application of molecular biology technology in the identification of counterfeit Chinese medicinal materials red deer antler is still in the preliminary research s

Method used

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  • Primer, probe, kit and method for identifying authenticity of cervus elaphus linnaeus
  • Primer, probe, kit and method for identifying authenticity of cervus elaphus linnaeus
  • Primer, probe, kit and method for identifying authenticity of cervus elaphus linnaeus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The inventors of the present invention cloned and sequenced the mitochondrial DNA D-loop region sequence of red deer for the first time by PCR.

[0031] This example is the sequence of the red deer mitochondrial DNA D-loop region obtained by PCR cloning and sequencing.

[0032] According to the difference of the mitochondrial DNA D-loop sequence in different cervids, the upstream and downstream primers were designed to amplify the red deer mitochondrial DNA D-loop sequence. The elk PCR product was purified and recovered according to the instructions of the Wizard GelExtraction Kit (Promega, USA). According to the instructions of the TaKaRa pMD19-T Vector 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...

Embodiment 2

[0047] In this embodiment, the extraction quality of the total DNA of the sample is tested by using a universal primer pair and a probe.

[0048] By detecting the sequence of the mitochondrial DNA D-loop region, the quality of the total DNA extraction from 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 react...

Embodiment 3

[0077] In this example, the specificity and sensitivity of the red deer primer pair and probe were verified through the following tests.

[0078] The specificity and detection sensitivity of red deer primer and probe combinations can be determined by detecting the mitochondrial DNA D-loop region sequence. The reaction system is: Fast Start Universal Probe MasterMix (Rox) 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.

[0079] The primers and probe sequences used to identify the authenticity of red deer antler are as follows: the primer sequences are SEQ ID No.1 and SEQ ID No.2, the probe sequence is SEQ ID No.3, and a fluorescence quencher is connected to the 3' end The 5' end of the group BHQ1 is connected with a fluorescent reporter group FAM.

[0080] The main detection instruments used:

[00...

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Abstract

The invention relates to a specific oligonucleotide primer, a probe and a real-time fluorescence polymerase chain reaction (PCR) detection method for identifying authenticity of cervus elaphus linnaeus. The method comprises the using of the specific oligonucleotide primer and the probe of the invention. The invention also relates to a PCR detection kit for identifying the authenticity of the cervus elaphus linnaeus. The kit comprises the specific oligonucleotide primer and the probe of the invention. The invention also relates to application of the specific oligonucleotide primer and the probe of the invention to the identification of the authenticity of the cervus elaphus linnaeus. By using the real-time fluorescence PCR detection method of the invention, the authenticity of the cervus elaphus linnaeus can be simply, quickly, specifically and flexibly identified.

Description

technical field [0001] The present invention belongs to the field of biotechnology, specifically, the present invention relates to specific oligonucleotide primers and probes for identifying the authenticity of red deer antler, a real-time fluorescent PCR detection method for identifying the authenticity of red deer antler, and the Application of specific oligonucleotide primers and probes or kit in identifying authenticity of red deer antler. Background technique [0002] Antler (Cornu Cervi Pantotrichum) is the young antler of deer (Cervus nippon Temminck) or red deer (Cervus elaphus Linnaeus) stag without ossification and dense hair. It is commonly called flower antler and red antler. Deer antler was first recorded in "Shen Nong's Materia Medica" and listed as a middle-grade product. It has the functions of strengthening kidney yang, benefiting essence and blood, and strengthening bones and muscles. It is one of the precious animal medicines in my country. There are many...

Claims

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Application Information

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