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Specific amplification primers of ganoderma mitochondrion rns gene and application of specific amplification primer

A technology for amplifying primers and mitochondria, which is applied in the field of genetic evolution, can solve problems such as homonyms and different names, homonyms and different names, and naming confusion of Ganoderma lucidum strains, and achieve the effects of simple operation, cost saving, and convenient use

Pending Publication Date: 2019-11-05
SAAS BIOTECH & NUCLEAR TECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, with the expansion of the cultivation scale, the nomenclature of ganoderma strains on the market is relatively confusing, and the phenomenon of the same name and different objects and the same object with different names is very prominent, which limits the further development and utilization of ganoderma resources.

Method used

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  • Specific amplification primers of ganoderma mitochondrion rns gene and application of specific amplification primer
  • Specific amplification primers of ganoderma mitochondrion rns gene and application of specific amplification primer
  • Specific amplification primers of ganoderma mitochondrion rns gene and application of specific amplification primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Establishment of Ganoderma lucidum rns gene sequence fragment database and primer design. Download all published mitochondrial rns gene sequences of Ganoderma species, and build a database of Ganoderma rns gene sequence fragments. Then, MEGA 6.06 software was used to compare the Ganoderma rns sequence to obtain the conserved segment of the Ganoderma rns gene. Design of universal primers for Ganoderma lucidum rns gene based on conserved segments. The designed primers should meet the following requirements: the length of the amplified fragment between the upstream and downstream primers is between 400-800bp, so that there are enough genetic loci for species differentiation and identification, and it can also ensure that the entire sequence can be measured in one sequencing reaction. After the total DNA of the species is extracted, there is no need to separate and extract the mitochondrial DNA of the species, which is simple to operate and saves cost.

[0029] In this ex...

Embodiment 2

[0031] Confirmation of primer specificity and validity of amplification method. In this example, through multiple single-factor optimization experiments, the PCR amplification conditions and reaction system of the Ganoderma lucidum rns gene were finally determined.

[0032] Among them, the optimal PCR amplification conditions are: 95°C for 5min; 94°C for 45s, 56°C for 45s, 72°C for 30s, 35 cycles; 72°C for 10min.

[0033] The 30 μL system includes: 15 μL of 2×PCR Mix, 1 μL of DNA template, 1 μL of upstream and downstream primers, ddH 2 O to make up to 30 μL.

[0034] The optimal PCR amplification conditions and systems provided in this example were used for PCR amplification to verify the specificity of the primers and the effectiveness of the method. The experimental method is as follows:

[0035] Five known species of Ganoderma lucidum were taken: red ganoderma, tropical ganoderma, pine fir ganoderma, white-fleshed ganoderma, and purple ganoderma. , USA) to extract total...

Embodiment 3

[0038] This example uses the primers provided in Example 1 and the PCR amplification conditions and system provided in Example 2 to identify species, providing further basis for the primers provided in the present invention and their applications.

[0039] In this example, in order to further verify the specificity of the primers, in this example, 15 species of Ganoderma lucidum, 3 species of Pleurotus, 4 species of Russula, and 9 species of Ascomycetes were amplified. Specifically include the following steps:

[0040]Firstly, DNA was extracted from 15 species of Ganoderma lucidum (known as Ganoderma lucidum, but the specific species was not known) and 16 species of non-Ganoderma lucidum. The fruiting bodies of each species were first fully ground with liquid nitrogen, and then the total DNA was extracted using the OMEGA Fungal DNA Extraction Kit (E.Z.N.A.TM Fungal DNA Kit, USA). Using the total DNA as a template (1 μL), amplification was performed using the PCR reaction cond...

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Abstract

The invention discloses specific amplification primers of a ganoderma mitochondrion rns gene and application of the specific amplification primers, and relates to the field of genetic evolution. The primers comprise an upstream primer and a downstream primer, a nucleotide sequence of the upstream primer is as shown in SEQ ID NO.1, and a nucleotide sequence of the downstream primer is as shown in SEQ ID NO.2. The primers can carry out specific amplification on ganoderma species at a time in large batches, interference of genes of animals, plants and microorganisms in samples or environment is effectively eliminated, and great convenience is provided for identification, variety breeding, phylogenetic analysis and strain monitoring of the ganoderma species. The magnitude of products amplifiedby the specific amplification primers is 650-750bp, sufficient genetic loci are provided for species distinguishing and identification, it can be guaranteed that all sequences can be measured throughone sequencing reaction, species mitochondria do not need to be independently separated and extracted, and the primers have the advantages of easy operation, cost saving and convenient using.

Description

technical field [0001] The invention relates to the field of genetic evolution, in particular to specific amplification primers for mitochondrial rns genes of Ganoderma lucidum and applications thereof. Background technique [0002] Mitochondria are the energy factories of eukaryotes, providing a source of energy (ATP) for most eukaryotes. Mitochondria contain their own genomes, reportedly derived from endosymbiotic bacteria. Mitochondrial genes have become a powerful tool for studying phylogeny and species classification due to their relatively small size, uniparental inheritance, evolution rate independent of nuclear genes, and a large number of homologous genes and molecular markers. [0003] Ganoderma lucidum (Amanita) is a general term for a class of fungi in the kingdom Fungi, Basidiomycota, Agaricaceae, Agaricales, Ganodermaceae, and Ganoderma genus, and is an important class of medicinal fungi. According to reports, the fruiting body of Ganoderma lucidum has the ef...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11C12Q1/6869G16B10/00C12R1/645
CPCC12Q1/6895C12Q1/6869G16B10/00C12Q2531/113
Inventor 李强黄文丽熊川金鑫李萍
Owner SAAS BIOTECH & NUCLEAR TECH RES INST
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