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Method for separating, screening and identifying earthworm gut culturable antibiotic resistant bacteria

A technology of antibiotic resistance, isolation and screening, applied in the field of soil biology, can solve the problems of colony screening, etc., and achieve the effect of easy control of the environment, simple operation and reliable operation method

Inactive Publication Date: 2019-01-11
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the microbial community obtained by this method can represent the microorganisms in the whole intestinal tract, the microbial community is located in Tris-EDTA buffer, which is very convenient for the extraction of total microbial DNA, but it has an impact on the screening of bacterial colonies

Method used

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  • Method for separating, screening and identifying earthworm gut culturable antibiotic resistant bacteria
  • Method for separating, screening and identifying earthworm gut culturable antibiotic resistant bacteria
  • Method for separating, screening and identifying earthworm gut culturable antibiotic resistant bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Method for the isolation, screening and identification of a single tetracycline-resistant bacterium in the intestinal tract of Eisenia chinensis

[0024] Samples were collected in an earthworm farm in Huaian City, Jiangsu Province. The farm used pig manure, cow dung, a small amount of wheat bran and mushroom residue as raw materials to raise earthworms, and the temperature was kept at about 25 °C. Select the red Eisensis with a body weight in the range of 0.3-0.5g and obvious rings (Eisenia foetida) As experimental objects, they were placed on ice packs and quickly transported back to the laboratory. Immediately transfer the returned earthworms to a Petri dish on ice, and let them stand for about 15 minutes to stop defecating. Wash the surface of the earthworm with neutral PBS buffer solution 2-3 times to remove impurities such as soil on the surface of the earthworm, and then put it back into the petri dish on ice. A series of optimizations have been carri...

Embodiment 2

[0032] Example 2 The method of isolation, screening and identification of sulfamethoxazole-resistant bacteria in Daping No. 2 intestinal tract

[0033] Samples were collected in an earthworm farm in Huaian City, Jiangsu Province. The farm used pig manure, cow dung, a small amount of wheat bran and mushroom residue as raw materials to raise earthworms, and the temperature was kept at about 25 °C. The Daping No. 2 with a weight in the range of 0.3-0.5g and an obvious ring was selected as the experimental object, and it was placed on an ice pack and quickly transported back to the laboratory. Immediately transfer the returned earthworms to a Petri dish on ice, and let them stand for about 15 minutes to stop defecation. Wash the surface of the earthworm with neutral PBS buffer solution 2-3 times to remove impurities such as soil on the surface of the earthworm, and then put it back into the petri dish on ice. Afterwards, 3 earthworms were placed in 50wt.% absolute ethanol. During...

Embodiment 3

[0035] Example 3 Method for Isolation, Screening and Identification of Tetracycline-Sulfadiazine Resistant Bacteria in the Intestines of the William Ringworm

[0036] Samples were collected in an earthworm farm in Huaian City, Jiangsu Province. The farm used pig manure, cow dung, a small amount of wheat bran and mushroom residue as raw materials to raise earthworms, and the temperature was kept at about 25 °C. Select a William's ringworm with a body weight in the range of 3-5g and obvious rings (Pheretima guillelmi) As experimental objects, they were placed on ice packs and quickly transported back to the laboratory. Immediately transfer the returned earthworms to a Petri dish on ice, and let them stand for about 15 minutes to stop defecation. Wash the surface of the earthworm with neutral PBS buffer solution 2-3 times to remove impurities such as soil on the surface of the earthworm, and then put it back into the petri dish on ice. Afterwards, three earthworms were placed ...

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Abstract

The invention discloses a method for separating, screening and identifying earthworm gut culturable antibiotic resistant bacteria. The method comprises the following steps: selecting fresh earthwormswhich have obvious clitellum and of which the weights are in a certain range, cleaning the body surface, performing anesthesia, dissecting, and taking intestinal contents; diluting the intestinal contents into a certain concentration gradient with sterile water, coating the diluents of different concentrations on an improved LB solid plate containing antibiotics of different types and contents, and culturing, screening and purifying the bacteria; culturing the purified bacterial colony in an improved LB liquid medium, and then extracting DNA by using a bacterial DNA extraction kit; performing16S rRNA amplification on the extracted DNA, and detecting the amplification product by using gel electrophoresis having the mass fraction of 2%; and performing sequencing analysis on the amplification product, and carrying out comparison on the NCBI after obtaining the sequence.

Description

technical field [0001] The invention belongs to the field of soil biology, and in particular relates to a method for isolating, screening and identifying antibiotic-resistant bacteria that can be cultivated in the intestinal tract of earthworms. Background technique [0002] As the invertebrate with the largest biomass in terrestrial ecosystems, earthworms can improve soil physical and chemical properties, participate in soil energy transfer and material cycle, and are called "soil ecosystem engineers". Earthworms can decompose and transform fermented organic waste in nature in their digestive system, and form vermicompost after excretion. Vermicompost is a kind of environment-friendly organic fertilizer resources. It has the advantages that ordinary organic fertilizers do not have, such as no odor residue after application, nutrients are more easily absorbed by crops, and soil-borne diseases are inhibited. The digestive system of earthworms plays a vital role in the form...

Claims

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Application Information

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IPC IPC(8): C12N1/02C12Q1/686C12Q1/04
CPCC12N1/02C12Q1/686C12Q2565/125C12Q2535/122
Inventor 孙明明晁会珍赵远超郑晓璇刘满强陈小云焦加国胡锋
Owner NANJING AGRICULTURAL UNIVERSITY
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