Characteristic nucleotide sequence as well as nucleic acid molecular probe and method for identifying ganoderma leucocontextum

A nucleotide sequence, white-fleshed Ganoderma lucidum technology, applied in microorganism-based methods, biochemical equipment and methods, microbial assay/inspection, etc. Needle and other problems, to achieve the effect of rapid and accurate identification, rapid identification, and good specificity

Inactive Publication Date: 2015-06-03
GUANGDONG INST OF MICROORGANISM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no nucleotide characteristic sequence, nucleic acid molecular probe and method for identifying Ganoderma lucidum

Method used

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  • Characteristic nucleotide sequence as well as nucleic acid molecular probe and method for identifying ganoderma leucocontextum
  • Characteristic nucleotide sequence as well as nucleic acid molecular probe and method for identifying ganoderma leucocontextum
  • Characteristic nucleotide sequence as well as nucleic acid molecular probe and method for identifying ganoderma leucocontextum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Obtaining the Characteristic Nucleotide Sequence of Ganoderma lucidum

[0025] 1. Extraction of ganoderma ganoderma genome DNA template:

[0026] Inoculate white-fleshed Ganoderma lucidum on PDA plates, culture in dark at 25°C for 7-10 days, collect mycelium, grind it into powder, and extract total DNA according to the instructions of Ezup Column Genomic DNA Extraction Kit (Fungi) to obtain white meat Genomic total DNA template of Ganoderma lucidum.

[0027] 2. rDNA ITS sequence amplification and sequencing of Ganoderma lucidum:

[0028] Using the total genomic DNA of Ganoderma lucidum as a template, the universal primer pair ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3' as primers, the reaction system is 50 μl total volume: 5 μl of the DNA template obtained in step 1, Primer (10μmol / L) 4μl*2, 2*PCRTaqmix 25μl, ddH 2 O 12 μl. PCR reaction conditions: 94°C for 5min; 94°C for 1min, 55°C for 45s, 72°C for 1min, 30cycle; 72°C for 8min. ...

Embodiment 2

[0029] Embodiment 2: Design of nucleic acid molecular probe of ganoderma lucidum

[0030] The characteristic nucleotide sequence of Ganoderma lucidum shown in SEQ ID NO.1 was compared and analyzed with the ITS sequences of Ganoderma lucidum and its related species in the GenBank database to determine the specific sites on the ITS sequence of Ganoderma lucidum. Using Primer in NCBI -BLAST (primer design and specificity testing tool) designed primers and verified the specificity of the primers in the GenBank database at the same time, and verified the specificity and accuracy of the identification through the PCR method experiment, and screened out a pair of white meat Ganoderma lucidum specific primers, BRLZ -F: 5'-AGATCTGCGAAGCGTGCT-3' and BRLZ-R: 5'-AGAGGAGCCGACCGACAG-3', such as figure 1 shown.

Embodiment 3

[0031] Example 3: Nucleic acid molecular probes are used as primers to carry out PCR amplification of Ganoderma lucidum

[0032] Extract the genomic DNA of Ganoderma leucocontextum, use it as a template, and use the nucleic acid molecular probes BRLZ-F and BRLZ-R of Ganoderma lucidum as primers for PCR amplification. The total volume of the PCR reaction system is 50 μl: 2*PCRTaqmix 25 μl, DNA Template 5 μl, ddH 2 O 12μl, primer (10μmol / L) 4μl*2. PCR reaction conditions: 94°C for 5min; 94°C for 1min, 56°C for 30s, 72°C for 1min, 30cycle; 72°C for 8min. The PCR products were band recovered and sequenced to obtain a sequence length of 348bp, such as figure 2 Shown, as shown in SEQ ID NO.2 of the sequence listing.

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Abstract

The invention discloses a characteristic nucleotide sequence, as well as a nucleic acid molecular probe and a method for identifying ganoderma leucocontextum. The characteristic nucleotide sequence is shown in SEQ ID NO.1, wherein the nucleic acid molecular probe comprises BRLZ-F:5'-AGATCTGCGAAGCGTGCT-3' and BRLZ-R:5'-AGAGGAGCCGACCGACAG-3'. The identifying method comprises the steps of identifying through a PCR method by taking the nucleic acid molecular probe BRLZ-F and BRLZ-R as primers. Based on the characteristic nucleotide sequence SEQ ID NO.1 of the ganoderma leucocontextum, the specific nucleic acid probe BRLZ-F and BRLZ-R are designed and taken as the primers, the PCR amplification is performed by taking the DNA of the ganoderma leucocontextum as a template and the rapid identification of the ganoderma leucocontextum is realized. The nucleic acid molecular probe provided by the invention is strong in selectivity, rapid and accurate in identifying method and good in specificity.

Description

technical field [0001] The invention relates to the field of using molecular biology methods to identify rare edible and medicinal fungus species, and in particular to a characteristic nucleotide sequence, nucleic acid molecular probe and identification method for identifying ganoderma lucidum. Background technique [0002] Ganoderma lucidum is a precious Chinese herbal medicine. Ganoderma lucidum has a medicinal history of more than 2,000 years in my country. Detoxification, prevention and treatment of cardiovascular system diseases, anti-aging, anti-neurasthenia, lowering blood sugar and blood pressure, anti-allergic and other effects. [0003] Ganoderma leucocontextum (Ganoderma leucocontextum T.H.Li, W.Q.Deng, Sheng H.Wu, Dong M.Wang&H.P.Hu) is a newly discovered new species of Ganoderma endemic to China. It was published in "Mycoscience" in 2014. The new species is Wild Ganoderma lucidum growing on Qinggang trees was collected by Hu Huiping, an assistant researcher at t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895
Inventor 黄龙花胡惠萍刘远超钟元茂黄志勇谭武平吴丽霞
Owner GUANGDONG INST OF MICROORGANISM
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