Method for quickly enriching, separating and detecting bacteria

A bacteria and enrichment technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, can solve the problems of complicated operation, complicated detection equipment, and residual magnetic beads affecting the test, etc., to slow down Settling speed, fast settling speed, effect of optimized mass fraction

Active Publication Date: 2016-12-07
CHONGQING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is mainly to provide a method for rapidly enriching and separating bacteria in view of the problems of complicated operation, residual magnetic beads affecting the test, and complicated subsequent detection equipment in the existing rapid detection of bacteria.

Method used

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  • Method for quickly enriching, separating and detecting bacteria
  • Method for quickly enriching, separating and detecting bacteria

Examples

Experimental program
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Effect test

Embodiment 1

[0029] This example is the rapid isolation and detection of Escherichia coli, the specific steps are as follows:

[0030] (1) Escherichia coli liquid preparation

[0031] Pick and inoculate Escherichia coli JM109 inoculation loop into 60mL sterilized LB liquid medium, incubate at 37°C for 12h, centrifuge at 8000rpm for 3min, remove the medium, resuspend in PBS, adjust the concentration of bacteria solution to 10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 CFU / mL, spare. Another 2 mL of PBS without bacteria solution was taken as a blank control.

[0032] (2) Magnetic bead enrichment

[0033] Take 2mL of Escherichia coli liquid of each concentration described in (1) and blank PBS into corresponding glass bottles, add 50μL of magnetic beads (1mg / mL) respectively, place on a shaker at 150rpm, mix and incubate at 37°C for 1h, and magnetically After enrichment for 3 minutes, the supernatant was removed and resuspended in 150 μL of PBS solution with a pH of 3.8.

[0034] (3) PEG ...

Embodiment 2

[0038] This example is the rapid isolation and detection of Staphylococcus aureus, the specific steps are as follows:

[0039] (1) Preparation of Staphylococcus aureus bacterial liquid

[0040]Pick and inoculate the Staphylococcus aureus inoculation loop into 60mL sterilized LB liquid medium, incubate at 37°C for 12h, centrifuge at 8000rpm for 3min, remove the medium, resuspend in PBS, and adjust the bacterial concentration to 10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 CFU / mL, spare. Another 2 mL of PBS without bacteria solution was taken as a blank control.

[0041] (2) Magnetic bead enrichment

[0042] Take 2mL of the Staphylococcus aureus bacterial solution of each concentration mentioned in (1) and blank PBS into corresponding glass bottles, add 100μL of magnetic beads (1mg / mL), place on a shaker at 150rpm, and incubate at 37°C for 1h. After magnetic enrichment for 3 min, the supernatant was removed and resuspended in 150 μL of PBS with a pH of 5.0.

[0043] (3) PE...

Embodiment 3

[0047] (1) Escherichia coli liquid preparation and magnetic bead enrichment

[0048] Carry out with reference to step (1), (2) in embodiment 1.

[0049] (2) Gelatin separation

[0050] Take 400 μL of gelatin (molecular weight: 10,000) with a mass fraction of 20% in a plastic separation tube with a diameter of 5 mm and a length of 3 cm, accurately pipette 150 μL of the sample in (2) above the gelatin, and place the separation tube vertically on the magnetic separation rack Put it aside for 10 minutes, and observe the separation result.

[0051] Measurement results: Observe the bottom of the separation tube. When using gelatin with a mass fraction of 20% to separate different concentrations of E. coli bacterial liquid, put the separation tube on the magnetic separation stand for 10 minutes, and the 2 ~10 7 Within the range of CFU / mL bacterial solution, with the increase of the concentration of the bacterial solution, the black precipitate separated at the bottom of the separa...

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Abstract

The invention discloses a method for quickly enriching, separating and detecting bacteria. The method comprises the following steps: (1) mixing a sample to be detected with magnetic beads, oscillation incubation, carrying out magnetic separation, and suspending the magnetic beads subjected to magnetic separation in a PBS (phosphate buffer solution) to obtain residual magnetic beads and a bacteria-adsorbed magnetic bead suspension; (2) adding prepared gel into a separator tube, putting the suspension obtained in the step (1) on the gel, and carrying out magnetic separation; and (3) after finishing the magnetic separation, visually inspecting the black precipitate on the bottom of the separator tube and carrying out qualitative analysis on the result, or establishing standard samples with different concentrations to obtain the corresponding relationship between the black precipitate quantity and standard sample concentration, comparing the quantity of black precipitate obtained by separating the sample to be detected with the corresponding relationship, and judging the quantity range of bacteria contained in the sample to be detected. The method can implement quick separation on bacteria in a complex sample, and can perform quick recognition and semiquantitative judgment on the sample with the bacteria content of 10<2> CFU / mL or above.

Description

technical field [0001] The invention belongs to the field of microbial analysis and detection, and in particular relates to a method for rapidly enriching, separating and detecting bacteria. Background technique [0002] Food safety is a major issue of great concern to all countries in the world, and pathogenic bacteria contamination is one of the main problems affecting food safety. According to WHO statistics, there are hundreds of millions of people suffering from food-borne diseases in the world, and there are hundreds of millions of diarrhea cases every year, resulting in the death of about 3 million children under the age of 5, of which about 70% are caused by food-borne pathogenic bacteria contamination of food , so the monitoring and detection of foodborne pathogenic bacteria has received widespread attention from the society. However, the samples for food safety monitoring have the characteristics of complex background matrix and low bacterial content, which brings...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04C12Q1/10C12Q1/14C12R1/19C12R1/445
Inventor 徐溢车玉兰王人杰崔飞云赵斌李泽全
Owner CHONGQING UNIV
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