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Extraction method of candida fungal biofilm extracellular matrix

A technology of biofilm and Candida, which is applied in the biological field and can solve the problems of EPS not being able to identify components, missing components, etc.

Inactive Publication Date: 2018-08-07
ANHUI UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, from a series of reports by this group, we can find that ① about 20-40% of the extracted EPS cannot identify its components; ② the extraction method used will miss the water-insoluble or slightly water-soluble components of EPS
Due to the lack of research data from other laboratories in this area, there are still many uncertainties about the chemical composition of EPS

Method used

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  • Extraction method of candida fungal biofilm extracellular matrix
  • Extraction method of candida fungal biofilm extracellular matrix
  • Extraction method of candida fungal biofilm extracellular matrix

Examples

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Embodiment 1

[0059] Embodiment 1: A kind of extraction method of candida fungus biofilm extracellular matrix

[0060] Step 1. First, soak each section of about 6cm grooved medical grade polyvinyl chloride (PVC) catheter piece in 75% ethanol overnight to keep it sterile, and then take it out and soak it in sterile water for use. After activating SC5314, adjust the bacterial concentration to 1×10 6 CFU / mL, place the catheter piece in the shaker (37°C 220rpm / min) and incubate for 4 hours to make the white nipple adhere to the catheter piece. After adhesion, the catheter pieces were taken out, and five catheter pieces were placed in a centrifuge tube containing 40 mL of 1640 medium in a centrifuge tube at a constant temperature of 37°C for 24 hours to form a biofilm. After 24 hours, use sterilized tweezers to gently take out the catheter piece, put it in a test tube filled with 6 mL of sterile water, and vortex for 1 min.

[0061] Step 2. Extraction of static SC5314 EPS by ion exchange resi...

Embodiment 2

[0072] Embodiment 2: A kind of extraction method of candida fungi biofilm extracellular matrix

[0073] Non-Candida albicans EPS extraction under static conditions.

[0074] Candida tropicalis (ATCC750), Candida glabrata (ATCC15126), Candida krusei (ATCC1182) were formed in static models. In the static model, the resuspended fungal cells were diluted to 4-6 × 10 in RPMI-1640 medium 6 CFU / mL used. PVC strips and diluted strains were co-incubated at 37 °C for 4 h as previously described to facilitate initial cell adhesion. Afterwards, the PVC catheter piece was taken out with sterile tweezers, washed gently with PBS to remove non-adherent cells, put into a 50 mL centrifuge tube, and incubated at 37°C for 20 h.

[0075] Detect the EPS production of water extraction method and cation exchange resin (pH5.8, pH6.8, pH7.8). Add 6 mL of NaH to the catheter piece after film formation 2 PO 4 -Na 2 HPO 4 Ionic buffer solution (PH5.8, PH6.8, PH7.8), vortex for 1min, mild and ...

Embodiment 3

[0080] Embodiment 3: A kind of extraction method of candida fungus biofilm extracellular matrix

[0081] A method for extracting the extracellular matrix of a Candida fungus biofilm, comprising the following steps:

[0082] The first step: first soak the catheter piece with ethanol to make it sterile, then take out the catheter piece and soak it in sterile water, then activate the bacteria of the genus Candida, and adjust the concentration of the bacteria solution to 1×10 6 CFU / mL;

[0083] The second step: place the catheter piece in the bacterial solution, and then incubate on a shaking table to make the bacteria adhere to the catheter piece, then take out the catheter piece and place it in a centrifuge containing 1640 medium Incubate the tube at a constant temperature of 37°C for 20-28 hours to form a biofilm;

[0084] Step 3: Take out the catheter piece obtained in the second step, and then put it in disodium hydrogen phosphate-sodium dihydrogen phosphate buffer or Tris-...

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Abstract

An extraction method of a candida fungal biofilm extracellular matrix includes: preparing a film forming guide tube piece, putting the film forming guide tube piece into a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution or a Tris-HCl buffer solution, and sequentially performing vortex vibration and ultrasonic treatment to obtain suspension liquid; subjecting the suspensionliquid and ion exchange resin to shaking table incubation in a container; using a 500mM NACl solution to elute the ion exchange resin obtained in the third step to obtain elution liquid, subjecting the elution liquid to shaking table action for 1.5-2.5 hours, filtering the elution liquid through a 0.22-micron microporous filter membrane, and centrifuging to obtain supernatant, namely candida fungal biofilm extracellular matrix extract. When the extraction method assisted by the ion exchange resin is applied to common candida fungal biofilm EPS (extracellular polymeric substances) extraction, high extraction efficiency can be achieved.

Description

technical field [0001] The invention relates to a method for extracting the extracellular matrix of a Candida fungus biofilm, and belongs to the field of biotechnology. Background technique [0002] Candida ( Candida , CA) is an opportunistic pathogenic fungus common on the surface of human mucosa. In recent years, the widespread application of broad-spectrum antibiotics, medical catheter intubation, invasive surgery, and radiation chemotherapy has caused the incidence of CA to increase year by year, and the clinical mortality rate is as high as 40-60%. One of the important reasons is that CA adhering to the surface of tissues or implantable materials can secrete abundant extracellular polymeric substances (EPS), and the biofilm composed of EPS and membrane bacteria wrapped in it ( biofilm) can protect CA from the attack of antifungal drugs and the recognition of the host immune system, among which EPS is considered to play a role in delaying the diffusion and penetration ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/16C12N1/00C12N13/00C12R1/72
CPCC12N1/005C12N1/16C12N13/00
Inventor 邵菁汪天明施高翔段强军吴大强笪文悦汪长中颜贵明
Owner ANHUI UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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