Method for extracting and enriching phage in soil

A bacteriophage and enrichment technology, applied in the field of soil biology, can solve the problems of low phage acquisition rate, time-consuming and labor-intensive operation, difficulty in passing through the membrane, etc., and achieves the effect of good operation prospect, simple operation, and simple operation method.

Inactive Publication Date: 2020-03-24
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

However, due to the large amount of sample required and the large amount of extracting solution used in soil, and because the SM buffer contains gelatin with a molecular weight of tens of thousands to hundreds of thousands, it will be difficult to pass through the membrane when used, and the operation is time-consuming and labor-intensive; and the phage enrichment process in the later stage In the 100kDa ultrafiltration pore size, a large number of soil phages will pass through the filter membrane, and the phage acquisition rate is low.
Therefore, both methods are not suitable for phage extraction and enrichment in soil

Method used

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  • Method for extracting and enriching phage in soil
  • Method for extracting and enriching phage in soil
  • Method for extracting and enriching phage in soil

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1 Method for Soil Phage Extraction and Enrichment in Grassland Soil

[0024] Grassland soil (a total of three samples, numbered Grassland-1, Grassland-2, and Grassland-3) was collected in Nanjing Agricultural University, and transported back to the laboratory at 4 °C. After removing impurities such as plant roots and stones in the soil, mix 300g of soil samples with 1L of 1% potassium citrate buffer solution. Each liter of potassium citrate buffer solution contains: 10g C 6 h 5 K 3 o 7 , 1.92 g Na 2 HPO 4 12H 2 O, 0.24g KH 2 PO 4 , pH=7, placed at 4°C for low-temperature incubation for 15 minutes to disperse the soil sample, and subjected it to short-term ultrasonic treatment in an ice-bath environment. shaken manually for 30 s; the total time of ultrasound was 3 minutes, and the ultrasound conditions were 100 W, 47 kHz. Since the soil texture is fine and difficult to centrifuge, centrifuge at 8000rpm for 10 minutes to obtain the supernatant, transfer the...

Embodiment 2

[0025] Example 2 The method for extracting and enriching soil phages from chromium-contaminated soil

[0026] The formulations of each reagent are the same as in Example 1. In a chromium slag contaminated site in Luzhou City, Sichuan Province (two samples in total, numbered Luzhou-1, Luzhou-2) and a chromium slag contaminated site in Zhangye City, Gansu Province (one sample in total, numbered Zhangye -1) Obtain chromium-contaminated soil, remove impurities from the contaminated soil, mix 300g of soil sample with 1L of 1% potassium citrate buffer, and place it at 4°C for 15 minutes to incubate the soil sample to disperse the soil sample, and place it in an ice bath Ultrasonic treatment in the environment, the ultrasonic process is as follows: after every 1 minute of ultrasonication, the extract was taken out of the ice bath and shaken manually for 30 s; the total ultrasonic time was 3 minutes, and the ultrasonic conditions were 100 W and 47 kHz. Centrifuge at 7000rpm for 10 min...

Embodiment 3

[0027] Example 3 Method for Extracting and Enriching Soil Phage from Pesticide Contaminated Soil

[0028] The formulations of the reagents are the same as in Example 1. Samples were collected at a pesticide-contaminated site in Qingyang Town, Nantong City, Jiangsu Province. The site was formerly a pesticide factory, and the soil in the site had varying degrees of pesticide pollution due to years of production. Collect the soil of plots with different degrees of pollution with less pollution (a total of six samples, numbered: Nantong-1, Nantong-2, Nantong-3, Nantong-4, Nantong-5, Nantong-6) at 4 °C Send back to lab. After removing impurities from the soil, mix 300g of the soil sample with 1L of 1% potassium citrate buffer, and incubate at 4°C for 15 minutes to disperse the soil sample, and then ultrasonicate it in an ice bath environment. The ultrasonic process is as follows: : After every 1 minute of ultrasonication, the extract should be taken out of the ice bath and shaken ...

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Abstract

The invention relates to a method for extracting and enriching phage in soil, which comprises the following steps: mixing fresh soil with a potassium citrate buffer solution according to a certain ratio, incubating the materials at low temperature for a period of time, and carrying out ultrasonic oscillation treatment on an extracting solution to extract the phage in soil from soil particles, centrifuging soil turbid liquid for multiple times to obtain a supernatant, sequentially filtering and degerming by using a sterilization filter membrane, reducing the volume of a filtrate by using a tangential flow filtration/ultrafiltration technology, and enriching bacteriophage; recovering bacteriophage in a polyethylene glycol precipitation manner, performing standing overnight at a low temperature, centrifuging the next day, dissolving the bacteriophage in a TE buffer solution, filtering the solution by using a filter membrane again for sterilization, and digesting impurities DNA and RNA ina sample by using nuclease; and finally performing subsequent DNA extraction, sample fixation and other operations on the obtained concentrated solution.

Description

technical field [0001] The invention belongs to the field of soil biology, in particular to a method for extracting and enriching phages in soil. Background technique [0002] Viruses that infect prokaryotic microorganisms are called phages, which are the most abundant organisms in the known natural environment; and because the number of prokaryotic microorganisms (bacteria and archaea) in soil is much greater than that of eukaryotes, phages are the most abundant organisms in the soil. major virus groups. Phages complete predation on host bacteria through five stages of adsorption, injection, proliferation (replication and biosynthesis), maturation (assembly) and release, and play an important role in soil: phages lyse bacteria, affect bacterial physiological metabolism, and thus affect The community structure and diversity of microorganisms in the ecosystem; the release of organic matter after bacterial lysis will affect the biogeochemical cycle of elements; bacterial DN...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12R1/92
CPCC12N7/00C12N2795/00051
Inventor 孙明明郑晓璇晁会珍王鑫伟胡锋
Owner NANJING AGRICULTURAL UNIVERSITY
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