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Characteristic Nucleotide Sequence, Nucleic Acid Molecular Probe and Method for Identifying Cantonese Cordyceps

A technology of nucleotide sequence and Cordyceps guangdong, which is applied in biochemical equipment and methods, measurement/inspection of microorganisms, DNA/RNA fragments, etc., can solve problems that have not yet been patented, and achieve good specificity, simple methods, and materials The effect of using less

Active Publication Date: 2011-12-21
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, patents have been obtained for the characteristic nucleotide sequences used to identify Cordyceps sinensis, Cordyceps japonicus, etc., but there is no patent for the characteristic nucleotide sequences related to the identification of Cordyceps guangdong

Method used

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  • Characteristic Nucleotide Sequence, Nucleic Acid Molecular Probe and Method for Identifying Cantonese Cordyceps
  • Characteristic Nucleotide Sequence, Nucleic Acid Molecular Probe and Method for Identifying Cantonese Cordyceps
  • Characteristic Nucleotide Sequence, Nucleic Acid Molecular Probe and Method for Identifying Cantonese Cordyceps

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Experimental program
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Embodiment 1

[0020] Extraction of Genomic DNA of Cordyceps guangdongensis and Acquisition of ITS rDNA Gene

[0021] Take 0.1g sample of Cordyceps guangdong and place it in a sterile mortar, pour liquid nitrogen into it and grind it quickly, add 600 μl of 2% (w / v) CTAB extraction buffer, which contains 2% (mass volume fraction) of SDS , continue to grind for a few seconds, then pour into a 1.5ml microcentrifuge tube, and keep warm in a water bath at 65°C for 1h. After cooling to room temperature, add an equal volume of saturated phenol:chloroform:isoamyl alcohol (volume ratio 25:24:1) solution to the 1.5ml microcentrifuge tube containing the sample, invert and mix for 2min, centrifuge at 12000rpm for 15min, and use 200μl Pipette the supernatant into a new centrifuge tube and discard the pellet. Add an equal volume of chloroform:isoamyl alcohol (volume ratio 24:1) solution to the supernatant, invert and mix for 2min, centrifuge at 12000rpm for 15min, and use a 200μl micropipette to carefull...

Embodiment 2

[0025] PCR Amplification of Cordyceps guangdong Specific Primers

[0026] According to the ITS rDNA gene sequence of Cordyceps guangdong (SEQ ID NO.1), a pair of sequences composed of 18-24 nucleotides, namely GDF and GDR, was designed by using the primer design software primer6. The sequences are as follows,

[0027] GDF:5`-CTGTTGGCATCTTCTGAGTCTC-3`;

[0028] GDR: 5`-GGTGCGAGGTTGTGCTACTA-3`.

[0029] Primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0030] Use the HS-taq Mix kit of Dongsheng Biotechnology Co., Ltd., its product number is P2081, to perform PCR reaction, using the genomic DNA of Cordyceps guangdong as a template, and the reaction system is 25 μL. Total volume: ddH 2 O 12.8 μL, 10 times buffer (Mg 2+ ) 8 μL, dNTP (2.5 mmol / L) 2 μL, GDF (10 μmol / L) 0.5 μL, GDR (10 μmol / L) 0.5 μL, HotStart Taq (5u / μL) 0.2 μL, template DNA 1 μL. The reaction program was pre-denaturation at 94°C for 5 minutes, 35 cycles at 94°C for 30s, 60°C for 20s, 72°C for ...

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Abstract

The invention discloses a characteristic nucleotide sequence, nucleic acid molecular probes and a method for identifying Cordyceps guangdongensis. The characteristic nucleotide sequence is shown as SEQ ID NO.1. The nucleic acid molecular probes comprise GDF of 5'-CTGTTGGCATCTTCTGAGTCTC-3' and GDR of 5'-GGTGCGAGGTTGTGCTACTA-3'. In the method, the nucleic acid molecular probes of GDF and GDR are used as polymerase chain reaction (PCR) primers, and a PCR method is used for identifying the Cordyceps guangdongensis; and steps are carried out according to a conventional PCR method. The GDF and the GDR are used as primers, the genome DNA of the Cordyceps guangdongensis is taken as a template, PCR is carried out according to the conventional PCR method, and the Cordyceps guangdongensis is amplified to a specific segment. Through experiments, only the Cordyceps guangdongensis can be amplified to the specific segment, while other Cordyceps samples such as Cordyceps sinensis, Cordyceps gunnii, Cordyceps militaris, supergene Cordyceps and Cordyceps brasiliensis are not amplified to any segment, so that the nucleic acid molecular probes of GDF and GDR have high specificity and can be used for quickly identifying truth and false of the Cordyceps guangdongensis (a fruit body and a mycelium) and related products.

Description

Technical field: [0001] The invention belongs to the technical field of using molecular biology methods to detect the authenticity of Chinese medicinal materials, in particular to a characteristic nucleotide sequence and a nucleic acid molecular probe and A method for identification of Cordyceps guangdongensis. Background technique: [0002] Cordyceps sinensis (Cordyceps sinensis) is a precious Chinese herbal medicine in China. It has the functions of protecting the lungs and kidneys, nourishing the essence and marrow, reducing phlegm and relieving cough. Regular consumption can promote digestion, regulate immune function, and enhance human resistance. The market demand is increasing day by day. However, with people's excessive collection, wild Cordyceps resources are becoming increasingly scarce and almost endangered, and the price has risen sharply, forming a vicious circle. The commercial production of Cordyceps sinensis fermented mycelium and Cordyceps militaris (myceli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 李挺宋斌李泰辉
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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