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Characteristic nucleotide sequence, nucleic acid molecular probes and method for identifying Cordyceps guangdongensis

A Guangdong Cordyceps, nucleic acid molecule technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as no patents yet, and achieve the effects of good specificity, simple method and short duration

Active Publication Date: 2013-11-13
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 probes and method for identifying Cordyceps guangdongensis
  • Characteristic nucleotide sequence, nucleic acid molecular probes and method for identifying Cordyceps guangdongensis
  • Characteristic nucleotide sequence, nucleic acid molecular probes and method for identifying Cordyceps guangdongensis

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Experimental program
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Effect test

Embodiment 1

[0020] Extraction of genomic DNA from Cordyceps guangdong and acquisition of ITS rDNA gene

[0021] Take 0.1 g of Cordyceps guangdong sample 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, pour it into a 1.5ml microcentrifuge tube, and keep it in a 65°C water bath for 1h. After cooling to room temperature, add an equal volume of saturated phenol:chloroform:isoamyl alcohol (volume ratio: 25:24:1) solution to a 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 2 minutes, centrifuge at 12,000 rpm for 15 minutes, and carefully remove the DNA-containing upper layer...

Embodiment 2

[0025] PCR amplification of specific primers for Cordyceps guangdong

[0026] According to the ITS rDNA gene sequence (SEQ ID NO.1) of Cordyceps sinensis in Guangdong, a pair of sequences consisting of 18-24 nucleotides, namely GDF and GDR, were designed using the primer design software primer6, and 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., the product number is P2081, to carry out the PCR reaction, using the genomic DNA of Cordyceps guangdong as the template, the reaction system is 25μL total volume: ddH 2 O 12.8 μL, 10x buffer (Mg 2+ ) 8μL, dNTP (2.5mmol / L) 2μL, GDF (10μmol / L) 0.5μL, GDR (10μmol / L) 0.5μL, HotStart Taq (5u / μL) 0.2μL, template DNA 1μL. The reaction program was pre-denaturation at 94 °C for 5 min, 35 cycles of 94 °C for 30 s, 60 °C for 20 s,...

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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, and particularly relates to characteristic nucleotide sequences and nucleic acid molecular probes for identifying Cordyceps guangdongensis T.H.Li, Q.Y Lin & B.Song. Methods for the identification of Cordyceps in Guangdong. Background technique: [0002] Cordyceps sinensis (Cordyceps sinensis) is a precious traditional Chinese medicinal material in China. It has the functions of protecting the lungs and kidneys, invigorating the essence and nourishing the marrow, resolving 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 the excessive collection of people, wild Cordyceps resources are becoming increasingly scarce, almost endangered, and prices have risen sharply, forming a vicious c...

Claims

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Application Information

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