Method for detecting bacteria by using protoplast through capillary electrophoresis

A technology of capillary electrophoresis and protoplasts, applied in the field of biological analysis, achieves the effects of high accuracy, simple preparation and operation, and low cost

Inactive Publication Date: 2013-09-25
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is of great theoretical and practical value to study the recognition of protoplasts by ssDNA or drugs, and then realize the detection of related bacteria. At present, there is no research on the application of capillary electrophoresis to detect the interaction between protoplasts and ssDNA or drugs to realize the detection of bacteria. method reporting

Method used

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  • Method for detecting bacteria by using protoplast through capillary electrophoresis
  • Method for detecting bacteria by using protoplast through capillary electrophoresis
  • Method for detecting bacteria by using protoplast through capillary electrophoresis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] cultivate bacteria:

[0059] (1) Inoculate Escherichia coli on the ultra-clean workbench, pick a single colony of Escherichia coli from the plate, inoculate it in LB liquid medium, and cultivate it on a shaking table for 12 hours at a temperature of 37°C and a rotation speed of 200 rpm to obtain cultured Good E. coli.

[0060] (2) Inoculate Lactobacillus acidophilus on the ultra-clean workbench, pick a single colony of Lactobacillus acidophilus from the plate, inoculate it in MRS liquid medium, and culture it statically for 48 hours at 37°C to obtain the cultured Lactobacillus acidophilus acid lactobacillus.

Embodiment 2

[0062] (1) Take 1 mL of Escherichia coli cultivated in Example 1, centrifuge at 3000r / min for 5min, discard the supernatant to obtain a precipitate, and centrifugally wash the precipitate with boric acid-borax buffer for 3 times, and add in the precipitate after washing. 100 μL of lysozyme with a concentration of 12 g / L, 50 μL of boric acid-borax buffer solution, and 50 μL of 2 mol / L sucrose solution were preheated at 37°C for 5 minutes to obtain mixed solution 1, which was placed in a centrifuge tube and placed in a 37°C Bath in the water bath for 25min, then add 0.05mol / L ethylenediaminetetraacetic acid (EDTA) 50μL to the mixed solution 1 to obtain the mixed solution 2, in which the concentration of EDTA is 0.01mol / L, and the mixed solution 2 is kept at 37℃ Submerged in a water bath for 15 minutes to obtain a protoplast mixed solution of Escherichia coli; the protoplast mixed solution of Escherichia coli was centrifuged at 3000r / min for 5 minutes, the supernatant was discarde...

Embodiment 3

[0069] (1) Get 2 mL of Lactobacillus acidophilus cultivated in Example 1, centrifuge at 3000r / min for 5min, discard the supernatant to obtain a precipitate, and the precipitate is washed 3 times by centrifugation with boric acid-borax buffer, and added to the precipitate after washing. Preheat 100 μL of lysozyme with a concentration of 24 g / L at 37°C for 5 minutes, 50 μL of boric acid-borax buffer solution and 50 μL of 2mol / L sucrose solution to obtain mixed solution 1, put mixed solution 1 in a centrifuge tube and put it at 37°C In a water bath for 30 minutes, add 50 μL of 0.05 mol / L EDTA to mixed solution 1 to obtain mixed solution 2, in which the concentration of EDTA is 0.01 mol / L, and continue to bathe mixed solution 2 at 37 ° C for 20 minutes to obtain The protoplast mixed solution of Lactobacillus acidophilus; the mixed solution of Lactobacillus acidophilus protoplast was centrifuged at 3000r / min for 5min, the supernatant was discarded, and the precipitation was centrifu...

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Abstract

The invention relates to a method for detecting bacteria by using protoplast through capillary electrophoresis. The method comprises the following steps of: (1) preparing the protoplast; (2) preparing a sample from bacteria protoplast to be detected and an ssDNA or medicament capillary electrophoresis buffer solution interacted with the protoplast, wherein in the sample, the concentration of protoplast suspension is 10<5>-10<9>CFU / mL, the concentration of the ssDNA solution is 2-20muM, and the concentration of the medicament solution is 0.5-10mmol / L; and (3) performing capillary electrophoresis detection on the sample prepared in the (2). According to the method, detection on the bacteria is realized by identifying the protoplast through the ssDNA or the medicament; the defects of few interaction sites and weak interaction force in the conventional method for identifying and detecting microbes by interaction of nucleic acid molecules and the microbes are overcome; and the method has the characteristics of accuracy, quickness, convenience and low cost.

Description

technical field [0001] The present invention relates to a kind of method that utilizes protoplast to detect bacterium by capillary electrophoresis, specifically, relate to a kind of application capillary electrophoresis to detect the interaction between protoplast and single-stranded DNA (single-stranded DNA, ssDNA) or medicine, realize ssDNA or The identification of protoplasts by drugs, and then the detection of bacteria, belongs to the field of biological analysis. Background technique [0002] At present, the methods of microbial detection and analysis mainly include physiological and biochemical identification, gas chromatography, immunology, molecular biology, Fourier transform infrared spectroscopy, three-dimensional fluorescence spectroscopy, and capillary electrophoresis. [0003] Among them, capillary electrophoresis (CE), also known as high performance capillary electrophoresis (HPCE), is a kind of separation channel with capillary, high-voltage DC electric field ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/447G01N21/33G01N21/64
Inventor 屈锋孟朝阳梅芳古力
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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