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Method for rapidly detecting bovine-derived shigella virulence by using defective caenorhabditis elegans

A technology of Caenorhabditis elegans and Shigella, applied in biochemical equipment and methods, preparations for in vivo experiments, measuring devices, etc., to save time and cost, reduce damage, and have broad application prospects

Pending Publication Date: 2021-03-30
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the pathogenic model of Caenorhabditis elegans infected by bovine Shigella has not been reported in China

Method used

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  • Method for rapidly detecting bovine-derived shigella virulence by using defective caenorhabditis elegans
  • Method for rapidly detecting bovine-derived shigella virulence by using defective caenorhabditis elegans
  • Method for rapidly detecting bovine-derived shigella virulence by using defective caenorhabditis elegans

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Experimental program
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Effect test

Embodiment 1

[0032] The processing of embodiment 1 Shigella

[0033] 1. Isolation and identification of bacteria: Bacteria isolated from bovine intestines in vitro were inoculated on MacConkey solid medium, and a colorless colony was isolated. Pick a single colony for Gram staining, microscopic examination, and inoculate on MacConkey medium, culture at 37°C for 16-18 hours, further purify and inoculate in LB liquid medium for re-cultivation.

[0034] 2. Bacterial DNA extraction: Fish the purified bacteria with a sterile inoculation loop, inoculate into MacConkey solid medium, incubate at 37°C for 16-18h, pick a single colony into LB broth, incubate at 37°C for 18h, follow Bacterial DNA Extraction Kit steps to extract DNA.

[0035] 3. PCR (polymerase chain reaction): use the extracted DNA as a template to amplify the 16S rRNA gene fragment, and the primer sequences are as follows:

[0036] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1);

[0037] 1492R:5'-GGCTACCTTGTTACGACTT-3' (SEQ ID NO...

Embodiment 2

[0044] Embodiment 2 nematode pathogenicity test

[0045] 1. Simultaneous treatment of Caenorhabditis elegans: Select an NGM plate with many adults and eggs and no mold contamination, and wash it repeatedly with M9 buffer to wash the nematodes and eggs from the OP50 bacterial lawn, and collect the sterilized plate. In a 15mL centrifuge tube, add M9 buffer solution to 10mL, centrifuge at 3000r / min for 1min, discard the supernatant, add 1mL nematode lysate, and vortex for 4min (the lysis time should not be too long, so as not to kill the eggs) , centrifuge at 3000r / min for 1min, and discard the supernatant. Add M9 buffer to make up to 10mL, centrifuge at 3000r / min for 1min, and repeat washing 3 times to remove the lysate. Finally, S basic solution was added to make the volume to 10 mL, and placed in a shaker at 20 °C and 150 r / min for 20 h. Then centrifuge at 3000r / min for 1min, discard the supernatant, transfer the L1 stage nematodes at the bottom of the centrifuge tube to a c...

Embodiment 3

[0052] Simultaneous treatment of Caenorhabditis elegans: Select an NGM plate with more adults and eggs and no mold contamination, wash it repeatedly with M9 buffer, wash the nematodes and eggs from the OP50 bacterial lawn, and collect them in a sterilized 15mL centrifuge In the tube, add M9 buffer solution to 10mL, centrifuge at 3000r / min for 1min, discard the supernatant, add 1mL of nematode lysate, vortex for 4min (the lysis time should not be too long, so as not to kill the eggs), 3000r Centrifuge for 1 min, and discard the supernatant. Add M9 buffer to make up to 10mL, centrifuge at 3000r / min for 1min, and repeat washing 3 times to remove the lysate. Finally, S basic solution was added to make the volume to 10 mL, and placed in a shaker at 20°C and 140 r / min for 22 hours. Then centrifuge at 1500r / min for 3min, discard the supernatant, transfer the L1 stage nematodes at the bottom of the centrifuge tube to a clean NGM plate, and place them in a biochemical incubator at 20°...

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Abstract

The invention discloses a method for rapidly detecting bovine-derived shigella virulence by using defective caenorhabditis elegans, relates to the technical field of biological models, and aims to rapidly and quantitatively detect the bovine-derived shigella virulence by utilizing sek-1, glp-1 gene defective caenorhabditis elegans more sensitive to pathogenic bacteria. The method has higher sensitivity to temperature, pathogenic bacteria and other factors, can efficiently detect the toxicity of shigella with high throughput, and can be used for researching the pathogenic mechanism of pathogenic bacteria and further screening antibacterial drugs, thereby providing a theoretical basis for clinical anti-infection treatment and medication of intestinal shigella.

Description

technical field [0001] The invention relates to the technical field of biological models, in particular to a method for rapidly detecting the virulence of bovine Shigella by using defective Caenorhabditis elegans. Background technique [0002] Shigella, also known as Shigella, can be divided into Shigella flexneri, Shigella sonnei, and Shigella dysenteriae. It is a Gram-negative bacillus with pili and no flagella. Colorless, translucent smooth colonies with a diameter of 2 mm grow on the culture medium. The pathogenic substances are mainly invasiveness and endotoxin, and some strains can also produce exotoxin, namely Shiga toxin (Stx). Stx belongs to a large family of bacterial toxins that cause severe gastrointestinal disease with high morbidity and mortality. Stx is one of the most powerful bacterial toxins known. The main types of Shiga toxins in disease are Stx1 and Stx2, which inhibit protein synthesis and eventually lead to apoptosis in affected tissues. Cattle and ...

Claims

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Application Information

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IPC IPC(8): C12Q1/10A61K49/00
CPCC12Q1/10A61K49/0008G01N2333/25
Inventor 彭昊李军白慧丽钟舒红李常挺吴翠兰陶立冯世文
Owner GUANGXI VETERINARY RES INST
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