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A primer and detection method for detecting arbuscular mycorrhizal fungi based on high-throughput sequencing

A technology of arbuscular mycorrhizal fungi and a detection method, applied in the field of primers and detection of arbuscular mycorrhizal fungi based on high-throughput sequencing, which can solve the problem of unsatisfactory detection of arbuscular mycorrhizal fungi, neglect, and inability to query sequences Detailed classification information and other issues to achieve the effect of high stability, less dosage and high efficiency

Active Publication Date: 2022-04-19
BAOTOU MEDICAL COLLEGE OF INNER MONGOLIA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current Illumina platform sequencing methods mostly focus on the ITS2 region, but because the primers for amplifying the ITS2 region are not specifically designed for arbuscular mycorrhizal fungi, the detection effect of arbuscular mycorrhizal fungi is not ideal
In addition, the 28SrRNA region of arbuscular mycorrhizal fungi has a better phylogenetic relationship than the ITS region. It is necessary to design a primer pair and comparison method for arbuscular mycorrhizal fungi based on the 28SrRNA region. In the current bioinformatics In the method, Cleandata is often used to directly compare with the database, so that the most similar results of the comparison are often sequences that are not annotated in detail, while sequences with clear annotations are ignored. For example, sequences with clear annotations in the database also display " In most cases, the detected sequences are compared in the database, and the sequences with the highest similarity are annotated as "unknown", and it is impossible to query the detailed classification information of the obtained sequences; therefore, it is necessary to establish a Annotating information sequences is a method of comparing sequences to improve the detection rate of arbuscular mycorrhizal fungi in samples and the accuracy of annotation

Method used

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  • A primer and detection method for detecting arbuscular mycorrhizal fungi based on high-throughput sequencing

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Experimental program
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Effect test

Embodiment 1

[0021] Detection of arbuscular mycorrhizal fungi species in clover plant tissue:

[0022] 1. DNA extraction

[0023] (1) Transfer 200 mg of clover plant tissue samples planted in the soil of Astragalus in Mongolia to a pre-cooled sterile mortar, immediately inject liquid nitrogen into the mortar, and use a mortar rod that has been pre-cooled with liquid nitrogen to saturate the tissue. Grind into powder, and immediately put it into a pre-cooled centrifuge tube and put it into a liquid nitrogen bottle;

[0024] (2) Add 1ml of CTAB extract solution preheated at 65°C to the centrifuge tube containing the sample, shake and mix on a vortex shaker to completely suspend the sample, and take a water bath at 65°C for 60 minutes;

[0025] (3) Shake 2 to 3 times during the water bath to ensure sufficient cracking. After taking it out, cool it to room temperature;

[0026] (4) Centrifuge at 8000rpm for 5min, and take the supernatant into a new 2.0ml sterile centrifuge tube;

[0027] (5...

Embodiment 2

[0114] Detection of arbuscular mycorrhizal fungi species in soil

[0115] 1. DNA extraction

[0116] Soil DNA extraction The OMEGA soil DNA extraction kit (catalog number D5625-02) was used to extract the genomic DNA of the soil. The specific methods are as follows:

[0117] (1) Weigh 500 mg of glass beads into a 15 ml centrifuge tube, add 0.5 g of soil samples from Astragalus mongolica, and then add 1 ml of Buffer SLX Mlus. Maximum speed vortex 3-5min;

[0118] (2) Add 100 μl DS Buffer, vortex and mix;

[0119] (3) Incubate at 70°C for 10 min, during which time, vortex gently to mix once. For samples with difficulty in lysis, the temperature can be adjusted to 90°C;

[0120] (4) Centrifuge at 3000 rpm for 3 min at room temperature. Transfer 800 μl of supernatant to a new 2 ml tube and add 270 μl BufferSP2. Vortex to mix the sample;

[0121] (5) Place on ice for 5 minutes, centrifuge at 13000ⅹg for 5 minutes at 4°C;

[0122] (6) Take the supernatant into a new 2ml cent...

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Abstract

The invention discloses a primer and a detection method for detecting arbuscular mycorrhizal fungi based on high-throughput sequencing. PCR amplification was performed after sequencing the universal primer adapter, and high-throughput sequencing was carried out after the PCR amplification product was purified. The measured original sequence was processed to obtain the Cleandata sequence, which corresponds to the reference sequence of the arbuscular mycorrhizal fungus 28SrRNA with clear annotation information. Blast comparison is carried out in the region, and by setting the similarity and coverage of the comparison, sequences with high similarity to the reference sequence are screened out; this method can sensitively detect arbuscular mycorrhizal fungal spores and plant spores in soil samples The mycelium of arbuscular mycorrhizal fungi in , and can enable arbuscular mycorrhizal fungi to be better annotated at the genus level, both for qualitative analysis and relative quantitative analysis.

Description

technical field [0001] The invention relates to a method and a special primer for identifying arbuscular mycorrhizal fungi in the field of biotechnology, and specifically designs a primer and a detection method for detecting arbuscular mycorrhizal fungi based on high-throughput sequencing. Background technique [0002] Arbuscular mycorrhizal fungi can form mutualistic symbionts "mycorrhizae" with most vascular plant roots. Host plants absorb mineral nutrients through arbuscular mycorrhizal fungi, which obtain photosynthetic products from plants. Because mycorrhizae can promote the growth and development of various plants, improve the ability of plants to resist adverse environments, and enhance the competitiveness of plants, it is a promising biological fertilizer. Arbuscular mycorrhizal fungi are a type of obligate biotrophic symbiotic bacteria, that is, they can grow, develop, reproduce and complete their life cycle normally only after establishing a symbiotic system with...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895
Inventor 王启刘广达
Owner BAOTOU MEDICAL COLLEGE OF INNER MONGOLIA UNIV OF SCI & TECH