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Analysis method for identifying bacteria and testing antibiotic sensitivity in biological sample

A technology of biological samples and bacteria, which is applied in the preparation of test samples, material analysis, material analysis by electromagnetic means, etc., can solve the problems of low content of metabolites, small size of a single bacterium, and sensitivity that cannot reach the sensitivity of a single bacterium analysis, etc. , to achieve the effect of fast speed and high sensitivity

Active Publication Date: 2021-11-12
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the small size of individual bacteria and the low content of metabolites have brought great challenges to the analysis of individual bacteria by mass spectrometry
At present, mass spectrometry is still based on culture in the detection of bacteria. After incubating individual bacteria into colonies, mass spectrometry is performed. The sensitivity of mass spectrometry analysis cannot reach the sensitivity of single bacteria analysis.

Method used

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  • Analysis method for identifying bacteria and testing antibiotic sensitivity in biological sample
  • Analysis method for identifying bacteria and testing antibiotic sensitivity in biological sample
  • Analysis method for identifying bacteria and testing antibiotic sensitivity in biological sample

Examples

Experimental program
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Embodiment 1

[0065] The detection and identification of a single live bacterium in the whole blood of embodiment 1 comprises the following steps:

[0066] (1) Capture localization of bacteria in whole blood:

[0067] Take 1 mL of bacteria-infected whole blood in a centrifuge tube, centrifuge at 3,000 rpm for 8 min, discard the blood cell pellet, centrifuge 500 μL of the supernatant containing bacteria at 10,000 rpm for 8 min, discard the supernatant, resuspend the pellet in 100 μL of deionized water, add 10 μL of 100 μM Stain with Acridine Orange stain for 15 minutes, centrifuge the stained solution at 10,000 rpm for 8 minutes, wash away the stain, and resuspend the pellet in 100 μL deionized water. Drop the resuspended bacterial solution on the poly-lysine glass slide and let it stand for 30 minutes to allow the bacteria to adsorb and fix on the poly-lysine glass slide, rinse with deionized water, and dry the poly-lysine slide at room temperature. Slide, observe the position of the bacte...

Embodiment 2

[0082] The detection and identification of a single live bacterium in the cell of embodiment 2 bacterial infection, comprises the following steps:

[0083] (1) Separation of bacteria in cells:

[0084] Add 100 μL of 10% saponin to 1 mL of RAW264.7 cell suspension infected with bacteria, lyse at 37°C for 5 min, centrifuge at 3000 rpm for 8 min, discard the precipitate, and keep the supernatant containing bacteria.

[0085] (2) Capture location of bacteria:

[0086] Add 10 μL of 100 μM Acridine Orange stain to the bacterial solution obtained in step (1) for staining for 15 minutes, centrifuge the stained solution at 10,000 rpm for 8 minutes, wash away the stain, and resuspend the precipitate in 100 μL of deionized water. Drop the resuspended bacterial solution on the poly-lysine glass slide and let it stand for 30 minutes to allow the bacteria to adsorb and fix on the poly-lysine glass slide, rinse with deionized water, and dry the poly-lysine slide at room temperature. slides...

Embodiment 3

[0099] The detection and identification of a single live bacterium in the bacterial infection tissue of embodiment 3 comprises the following steps:

[0100] (1) Isolation of bacteria from infected tissue:

[0101] Wipe the infected tissue with a sterile cotton swab, put the swab into 1mL deionized water to elute the bacteria, and keep the eluate containing the bacteria.

[0102] (2) Capture location of bacteria:

[0103]Add 10 μL of 1 mM Acridine Orange stain to the bacterial solution obtained in step (1) for staining for 15 minutes, centrifuge the stained solution at 10,000 rpm for 8 minutes, wash off the stain, and resuspend the precipitate in 100 μL of deionized water. Drop the resuspended bacterial solution on the poly-lysine glass slide and let it stand for 30 minutes to allow the bacteria to adsorb and fix on the poly-lysine glass slide, rinse with deionized water, and dry the poly-lysine slide at room temperature. slides, and observe the location of the bacteria under...

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Abstract

The invention discloses a method for identifying bacteria in a biological sample and a method for testing antibiotic sensitivity of the bacteria in the biological sample. The method for identifying the bacteria in the biological sample comprises the following steps: (1) centrifugally separating the bacteria in the biological sample; (2) dyeing the bacteria obtained in the step (1); (3) fixing the bacteria dyed in the step (2) through an electrostatic adsorption effect; (4) after an extraction solution is injected into the tip of a capillary tube, extracting metabolite of single live bacteria in an in-situ extraction manner; and (5) inserting an electrode into the capillary tube obtained in the step (4), applying voltage, and carrying out mass spectrometry. The method disclosed by the invention does not need to depend on incubation, can realize bacterial species identification and antibiotic sensitivity test in an initial bacterial infection biological sample, has the advantages of rapidness, high sensitivity, non-targeted analysis and the like, and has a good application prospect.

Description

technical field [0001] The present invention relates to the field of biology. Specifically, the present invention relates to analytical methods for bacterial identification and antibiotic susceptibility testing in biological samples. Background technique [0002] The detection or identification of bacteria is required in many different fields, and rapid and accurate bacterial identification is crucial in fields such as medicine and biology. Some biological samples have low bacterial content in the early stage of bacterial infection. For example, in the early stage of blood infection, there are only 1-1000 bacteria per milliliter of blood, and with the overuse and abuse of antibiotics, more and more bacteria mutate, which has great impact on Rapid and accurate bacterial detection presents even more stringent requirements. [0003] The culture method is currently the most commonly used method for the detection of bacteria in biological samples. The density of bacteria is inc...

Claims

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Application Information

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IPC IPC(8): G01N27/62G01N1/28G01N1/30G01N1/34
CPCG01N27/62G01N1/28G01N1/30G01N1/34
Inventor 黄光明詹柳娟侯壮豪
Owner UNIV OF SCI & TECH OF CHINA
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