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A kind of detection method of exopolysaccharide of aspergillus fumigatus biofilm

An extracellular polysaccharide and detection method technology, which is applied in the direction of chemical reaction of materials for analysis, and material analysis by observing the effect on chemical indicators, can solve the problems of high requirements for instruments and equipment, high price, and cumbersomeness. Achieve the effect of low equipment requirements, fast detection and low price

Active Publication Date: 2016-06-15
GUANGXI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, scientific experimental research often requires repeated observations of the changes in biofilms, but currently recognized and commonly used morphological observation methods such as scanning electron microscope (scanning electron microscope, SEM) and laser confocal microscope (lasers scanning confocal microscope, CLSM) have equipment The requirements are high and the price is expensive. The scanning electron microscope detection sample process has to go through cumbersome steps such as material collection-rinsing-fixation-rinsing-post-fixation-dehydration-drying-coating-scanning, etc., which is not easy to popularize and is difficult to promote clinically. It is of great significance to quickly, easily and reliably observe the morphology of fungal biofilm
Wang Yuan and others first discovered that Alcian blue Congo red staining can be applied to bacterial extracellular sugar staining. Now this method has been widely used in the study of bacterial biofilms. However, due to the different characteristics of bacteria and fungi, the characteristics of the biofilms formed by them It is not exactly the same. Whether the Alcian blue Congo red staining method can be applied to the exopolysaccharide staining of Aspergillus fumigatus biofilm has not been reported.
There is an urgent need for a rapid and accurate method for detecting the morphology of fungal biofilms clinically, which has become a major problem that limits the further development of fungal biofilm detection

Method used

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  • A kind of detection method of exopolysaccharide of aspergillus fumigatus biofilm
  • A kind of detection method of exopolysaccharide of aspergillus fumigatus biofilm

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] 1. Carrier: Glass cell slide, 13 mm in diameter (Jiangsu Haimen Shengbang Experimental Equipment Co., Ltd.).

[0042] 2. Strains: From September 2012 to May 2013, 30 strains of Aspergillus fumigatus were collected in the Bacteria Laboratory of the Laboratory Department of the First Affiliated Hospital of Guangxi Medical University. After 96-well plate film formation, the film-forming ability was compared by crystal violet semi-quantitative method The strongest strain was used as the test strain.

[0043] 3. Test reagents and equipment: Alixin blue 8GX (Shanghai Yuanye Biotechnology Co., Ltd.), Congo red (Tianjin Guangfu Fine Chemical Research Institute), PDA (Beijing Luqiao), MOPS (Sigma), RPMI-1640 powder (Gibco ), scanning electron microscope SEM (S-3400N, Hitachi, Japan), photomicrograph system (IMT-2, OLYMPAS, Japan), ordinary optical microscope (BH22, OLYMPAS, Japan).

[0044] 4. Preparation of Aspergillus fumigatus biofilm carrier Sterilized glass cell slides (13 m...

Embodiment 2

[0058] Example 2 Alcian Blue Congo Red Staining Results and Scanning Electron Microscope Results

[0059] The Aspergillus fumigatus biofilm carrier prepared in Example 1 was taken out at 8, 12, 16, 20, 24, and 48 hours, 2 each time, and stained with Alcian blue Congo red, and then magnified 200 times under an ordinary optical microscope. observation, the result is as figure 1 As shown, A: Part of the spores germinated at 8 hours; B: Mycelia elongated at 12 hours; C: Mycelia continued to grow at 16 hours, and a little dark red exopolysaccharide began to surround the blue mycelium; D: At 20 hours There are more hyphae, and exopolysaccharides have been connected between hyphae and hyphae; E: Mycelium has formed a three-dimensional structure at 24 hours, and the surface of the carrier is basically covered with exopolysaccharides, only a few hyphae on the top are not covered with extracellular polysaccharides. The exopolysaccharides were completely covered and turned blue; F: Myce...

Embodiment 3

[0062] 1. Carrier: Glass cell slide, 15mm in diameter (Jiangsu Haimen Shengbang Experimental Equipment Co., Ltd.).

[0063] 2. Strains: From December 2012 to July 2013, 30 strains of Aspergillus fumigatus were collected from the Bacteria Laboratory of the Laboratory Department of the First Affiliated Hospital of Guangxi Medical University, and the film-forming ability was compared by semi-quantitative crystal violet method after 96-well plate film formation The strongest strain was used as the test strain.

[0064] 3. The test reagents and equipment are the same as in Example 1.

[0065] 4. Preparation of Aspergillus fumigatus biofilm carrier Sterilized glass cell slides (15 mm in diameter) were used as carriers. Inoculate the tested Aspergillus fumigatus strain on a potato dextrose agar plate and culture it at 35°C. After 5 days of activation, rinse the surface of the plate with 10ml of phosphate buffer containing Tween 20 to collect spores, and resuspend the spores in 25ml ...

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Abstract

The invention discloses a detection method of exopolysaccharides of an aspergillus fumigatus biological membrane. The detection method comprises the following steps of preparing a carrier, collecting bacterial strains, preparing an aspergillus fumigatus biological membrane carrier, implementing an alcian blue Congo red staining method and a bacterial strain observation method; placing aspergillus fumigatus with the concentration being 1-5*10<5> spores / ml<-1> in a sterile glass slide of cells, standing the aspergillus fumigatus in a biochemical incubator at 35-37 DEG C for cultivating, respectively compositing the exopolysaccharides of a dye cell by utilizing the alcian blue Congo red staining at the different times during cultivation, and observing the change of the exopolysaccharides of the aspergillus fumigatus biological membrane. The detection method of the exopolysaccharides of the aspergillus fumigatus biological membrane, provided by the invention, has the advantages that the experiment operation is simple, the result is rapidly obtained, the repeatability is good, and the detection method is suitable for clinical examination and laboratory investigation.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a method for detecting aspergillus fumigatus biofilm exopolysaccharide by using an alcian blue congo red staining method. Background technique [0002] In recent years, with the increase in patients with malignant tumors, HIV, and organ transplants, Aspergillus fumigatus infection has shown a rapid development trend. The infection rate is second only to Candida albicans, and it has become the main cause of death in immunocompromised patients. The mortality rate of invasive aspergillosis (IA) caused by Aspergillus fumigatus reaches 30-100%, and drug resistance is an important reason for treatment failure. A clinical study tested the Aspergillus fumigatus strains of 1385 IA patients and found that azole The mortality rate of patients with drug-resistant IA was 88%, while the mortality rate of those infected with sensitive strains was 48%. In vivo and in vitro studies ...

Claims

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Application Information

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IPC IPC(8): G01N21/78
Inventor 陈一强黄宏孔晋亮
Owner GUANGXI MEDICAL UNIVERSITY