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Characteristic nucleotide sequence, nucleic acid molecule probes, kit and method for identifying ganoderma strain GIMS1524

A GIMS1524, nucleotide sequence technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., to achieve the effects of good specificity, rapid and accurate identification methods, and strong specificity

Inactive Publication Date: 2019-04-12
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some nucleotide characteristic sequences used to identify fungal strains have been patented, but because the wild Ganoderma lucidum GIMS1524 is a high-quality product obtained by the research team after 6 years of investigation, collection and research on wild macrofungus resources in various nature reserves across the country. strains, and the application of ISSR molecular markers to find the specific bands of the target strain, and through cloning sequencing, specific primer design verification, so as to obtain the application of the method of strain-specific specific probes on Ganoderma lucidum, has not been reported

Method used

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  • Characteristic nucleotide sequence, nucleic acid molecule probes, kit and method for identifying ganoderma strain GIMS1524
  • Characteristic nucleotide sequence, nucleic acid molecule probes, kit and method for identifying ganoderma strain GIMS1524
  • Characteristic nucleotide sequence, nucleic acid molecule probes, kit and method for identifying ganoderma strain GIMS1524

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Obtaining the characteristic nucleotide sequence of Ganoderma lucidum strain GIMS1524

[0027] 1. Extraction of Ganoderma lucidum genomic DNA template

[0028] Ganoderma lucidum was inoculated on a PDA plate, cultured in the dark at 25°C for 7-10 days, the mycelium was collected, ground into powder, and the total DNA was extracted according to the instructions of Magen's HiPure Fungal DNA Kit II Fungal DNA Extraction Kit. Genomic total DNA of Ganoderma lucidum was obtained.

[0029] 2. Specific band exploration of wild Ganoderma lucidum GIMS1524

[0030] Using the total genomic DNA of wild Ganoderma lucidum GIMS1524 and 21 strains of Ganoderma lucidum (including 1 wild strain (strain M150311) and 20 marketed strains (strains MC-GL-0043 to MC-GL-0062)) as templates, more than 100 ISSR primers were used as primers.

[0031] The total volume of the reaction system is 50 μL: DNA template 5 μL, primer (10 μmol / L) 4 μL×2, 2×PCR Taqmix 25 μL, ddH 2O 12 μL. PCR r...

Embodiment 2

[0034] Example 2 Design and Verification of Ganoderma Lucidum Strain GIMS1524 Nucleic Acid Molecular Probe

[0035] (1) Design of nucleic acid molecular probes

[0036] Using primer premier 5.0 software, primers were designed for the characteristic nucleotide sequence of wild Ganoderma lucidum GIMS1524, and specific primer pairs (nucleic acid molecular probes) were obtained, respectively GIM24-F:5'-CCGTCCTCTTCGGTATGTC-3'; GIM24-R: 5'-GTGGTCTGTGTATTGTTTG-3'. The design sites of the specific primers are shown in figure 2 .

[0037] (2) Verification of specific primer pairs

[0038] Genomic DNA of wild Ganoderma lucidum GIMS1524 was used as a template, GIM24-F: 5'-CCGTCCTCTTCGGTATGTC-3' and GIM24R: 5'-GTGGTCTGTGTATTGTTTG-3' were used as primers, and the reaction system was 50 μL. Total volume: 5 μL of DNA template, primers (10 μmol / L) 4μL×2, 2×PCR Taqmix 25μL, ddH 2 O 12 μL. PCR reaction conditions: 94°C for 5min; 94°C for 45s, 49°C for 30s, 72°C for 45s, 35cycle; 72°C fo...

Embodiment 3

[0039] Example 3 Identification of Ganoderma lucidum strain GIMS1524

[0040] Genomic DNA of wild Ganoderma lucidum GIMS1524 and 23 strains of Ganoderma lucidum (including 2 wild strains (strain M150311 and strain W140201) and 21 marketed strains (strain MC-GL-0043 to strain MC-GL-0063)) were used as templates, Using GIM24-F: 5'-CCGTCCTCTTCGGTATGTC-3' and GIM24-R: 5'-GTGGTCTGTGT ATTGTTTG-3' as primers, perform PCR amplification according to the reaction conditions in Example 2, and then use 1% agarose gel electrophoresis Imaging, when a target band with a size of about 420bp appears on the electropherogram, it means that the sample is wild Ganoderma lucidum GIMS1524, otherwise it is not. see results Figure 4 .

[0041] Depend on Figure 4 It can be seen that wild Ganoderma lucidum GIMS1524 can amplify a specific fragment with a size of about 420bp, while other samples of Ganoderma lucidum do not amplify any fragment, which shows that the nucleic acid molecular probe of the...

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Abstract

The invention discloses a characteristic nucleotide sequence, nucleic acid molecule probes, a kit and a method for identifying a ganoderma strain GIMS1524. The characteristic nucleotide sequence is asshown in SEQ ID NO.1. The nucleic acid molecule probes include GIM24-F: 5'-CCGTCCTCTTCGGTATGTC-3'; and GIM24-R: 5'-GTGGTCTGTGTATTGTTTG-3'. The identification method uses the nucleic acid molecular probes GIM24-F and GIM24-R as PCR primers, and performs identification by a PCR method. The method of the invention can achieve rapid identification of the ganoderma strain GIMS1524. The nucleic acid molecule probes of the invention have strong specificity, and the identification method is rapid and accurate, and good in specificity.

Description

technical field [0001] The invention relates to the technical field of using molecular biology methods to identify rare edible and medicinal fungi, in particular to a characteristic nucleotide sequence, nucleic acid molecular probe, kit and method for identifying ganoderma lucidum strain GIMS1524. Background technique [0002] Ganoderma lucidum (red lucidum) is a precious Chinese herbal medicine. Ganoderma lucidum has a medicinal history of more than 2,000 years in my country. , liver protection and detoxification, prevention and treatment of cardiovascular system diseases, anti-aging, anti-neurasthenia, lowering blood sugar and blood pressure, anti-allergy and other effects. [0003] Wild Ganoderma lucidum GIMS1524 has good and stable cultivation properties, high biotransformation rate, and the content of active ingredients such as polysaccharides is also significantly higher than that of red lucidum circulating in the market, which has good development and utilization value...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/04C12N15/11
CPCC12Q1/6895
Inventor 黄龙花胡惠萍谢意珍刘远超莫伟鹏梁晓薇
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY