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A high-throughput method for absolute quantification of soil bacteria

An absolute quantitative, high-throughput technology, applied in the field of soil microorganisms, to achieve simple, reliable cross-sample, relatively accurate and reliable results

Active Publication Date: 2018-10-02
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the plate culture counting method may meet the requirements of obtaining information on the types and quantities of microorganisms at the same time, but only 1%-3% of microorganisms in the environment can be cultivated by this technology; high-throughput sequencing technology can meet the requirements of obtaining a large number of microbial community structures Requirements for taxonomic information, but limited to characterizing species content in terms of relative abundance

Method used

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  • A high-throughput method for absolute quantification of soil bacteria
  • A high-throughput method for absolute quantification of soil bacteria
  • A high-throughput method for absolute quantification of soil bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1, the absolute quantification of the internal standard bacterial strain HTAQ-GFP is carried out by using the fluorescence microscope counting method and the plate culture counting method, and the following steps are carried out in sequence:

[0060] 1) Preparation of 2-fold serial dilution of bacterial suspension:

[0061] Pipette 500 μL of the mother solution of the above-mentioned internal standard strain HTAQ-GFP bacteria suspension into a 1.5 mL sterilized centrifuge tube, add 500 μL of sterilized water and mix well by pipetting to make a 2-fold diluted bacterial suspension. Draw 500 μL from the 2-fold diluted bacterial suspension into a 1.5mL sterilized centrifuge tube, add 500 μL sterilized water, blow and mix well to make a 4-fold diluted bacterial suspension; and so on, make a 2-fold gradient diluted bacterial suspension solution (the mother solution is diluted 2, 4, 8, 16, 32, 64, 128 times).

[0062] 2) Fluorescence microscope counting method:

...

Embodiment 2

[0071] Embodiment 2, add internal standard bacterial strain HTAQ-GFP, obtain soil bacteria absolute content (total amount and each classification level content)

[0072] 1) Add the internal standard strain to the sample:

[0073] Draw 200μL of the 10-fold diluted strain HTAQ-GFP bacterial suspension mother solution into a beaker containing 10g soil sample (dry soil), stir and mix for 5min under ice bath conditions, and obtain a final concentration of strain HTAQ-GFP of 10 6 cells g -1 Left and right soil samples. The soil samples added with equal volume of sterile water were used as control treatment.

[0074] 2) Extract soil DNA:

[0075] Weigh 0.25g of the soil sample obtained in step 1) to extract soil DNA. This example uses MoBio DNA extraction kit, the operation steps follow the standard procedure of the kit.

[0076] 3) High-throughput sequencing:

[0077] The soil sample DNA was subjected to high-throughput sequencing of bacterial 16S rRNA gene amplicons (Illumin...

Embodiment 3

[0082] Embodiment 3, the high-throughput absolute quantification method of soil bacteria is applied to the soil sample treated with sodium azide, and the following steps are carried out in sequence:

[0083] 1) Add 0.1% sodium azide (w / w) to the soil, culture at 28°C for 14 days, and take 10 g of soil samples from 0 and 14 days of culture for subsequent addition of internal standard strain HTAQ-GFP .

[0084] 2) Add the internal standard strain to the sample:

[0085] Draw 200μL of the 10-fold diluted strain HTAQ-GFP bacterial suspension mother solution into a beaker containing 10g soil sample (dry soil), stir and mix for 5min under ice bath conditions, and obtain a final concentration of strain HTAQ-GFP of 10 6 cells g -1 Left and right soil samples.

[0086] 3) Extract soil DNA:

[0087] Weigh 0.25g of the soil sample obtained in step 2) to extract soil DNA. This example uses MoBio DNA extraction kit, the operation steps follow the standard procedure of the kit.

[00...

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Abstract

The invention discloses a high-throughput absolute quantification method for soil bacteria. The high-throughput absolute quantification method includes steps of whirling and re-suspending E. coli HTAQ-GFP thalli in sterile water and regulating bacteria suspension in ultraviolet spectrophotometers OD <600nm=1.0>; 2), absolutely quantifying internal standard strains HTAQ-GFP and acquiring the absolute content of internal standard strains E. coli HTAQ-GFP in bacteria solution; 3), adding the bacteria solution with the internal standard strains HTAQ-GFP to soil samples and uniformly stirring and mixing the bacteria solution and the soil samples with one another; 4), extracting soil DNA (deoxyribonucleic acid) and carrying out high-throughput sequencing on amplicons of 16S rRNA [16S ribosomal RNA (ribonucleic acid)] genes to obtain classification composition information of bacterial community structures and corresponding relative abundance; 5), acquiring the total quantities of native bacteria in soil and the absolute contents of various classification units according to the absolute contents of the internal standard strains HTAQ-GFP and the relative abundance of Escherichia in high-throughput sequencing results.

Description

technical field [0001] The invention belongs to the field of soil microorganisms, and relates to a high-throughput absolute quantitative technology of soil bacteria integrating a high-throughput sequencing platform and a bacterial internal standard method. Background technique [0002] There are a large number and various types of microorganisms in the soil, and they play an indispensable role in the earth's material cycle, industrial and agricultural production, and even medical fields. Studying the succession process of microbial community structure in a specific ecological environment and obtaining information on its species and quantity changes are of great significance for further elucidating and utilizing microbial biochemical mechanisms and protecting microbial resources and diversity. [0003] Since Leeuwenhoek first observed the existence of microorganisms through a microscope, various methods for exploring the microscopic world of microorganisms have emerged. Exis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869C12Q1/06
CPCC12Q1/6869C12Q2535/122C12Q2545/114
Inventor 汪海珍杨黎楼骏徐建明
Owner ZHEJIANG UNIV
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