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Immunomagnetic bead and method for rapidly detecting Shewanella oneidensis

A technology of oneida Shewanella and immunomagnetic beads, which is applied in the field of microbial detection, can solve the problems of an immunomagnetic separation and detection method without Oneida Shewanella, and achieves convenient detection process, good specificity, Good suspension effect

Active Publication Date: 2014-07-30
GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A search of existing technologies found that there is currently no immunomagnetic separation detection method for Shewanella oneidae

Method used

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  • Immunomagnetic bead and method for rapidly detecting Shewanella oneidensis
  • Immunomagnetic bead and method for rapidly detecting Shewanella oneidensis
  • Immunomagnetic bead and method for rapidly detecting Shewanella oneidensis

Examples

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Effect test

preparation example Construction

[0036] Preparation of immunomagnetic beads

[0037] (1) Preparation of immune antigen: the immune antigen is prepared by inactivating Shewanella Oneida liquid with 1% formalin;

[0038] (2) Preparation of Oneida Shewanella-specific antibody: the Oneida Shewanella-specific antibody was obtained by immunizing New Zealand white rabbits with inactivated immune antigens to obtain antiserum, and a two-step ammonium sulfate salting-out method was adopted Purified antiserum to obtain Shewanella Oneida-specific antibodies;

[0039] (3) Preparation of immunomagnetic beads: the immunomagnetic beads are prepared by coupling purified Shewanella Oneida-specific antibodies and activated carboxylated polystyrene magnetic microspheres by shaking and incubating;

[0040] Method for Rapid Detection of Shewanella Oneida

[0041] 1) Add the immunomagnetic beads prepared above to the sample solution to be tested, shake and react at room temperature (18-27°C) for 10-70 minutes, so that the ant...

Embodiment 1

[0055] Example 1: Preparation of immunomagnetic beads

[0056] (1) Preparation of Shewanella Oneida-specific antibody

[0057] The glycerin preservation solution of Shewanella oneida was inoculated in a 500ml Erlenmeyer flask at a ratio of 1:100, and cultured in a constant temperature shaker at a temperature of 30°C and a rotation speed of 160rpm for 16h. Centrifuge the cultured bacteria solution at 5000g for 20min, collect the precipitate, wash 3 times with PBS, and adjust the concentration of the bacterial solution to 10 with PBS. 9 cfu / mL. After inactivating the bacterial suspension with 1% formalin for 24 hours, wash it 3 times with PBS, and adjust the concentration of the bacterial solution to 10 8 cfu / mL, and fully emulsified with Freund's adjuvant according to 1:1 (V / V) to prepare the immune antigen. The prepared antigen was used to immunize New Zealand white rabbits, 30 days after the first immunization, and then immunized once every 10 days, for a total of 4 immu...

Embodiment 2

[0060] Embodiment 2: the method for rapidly detecting Shewanella Oneida

[0061] 1) Add 10-50 immunomagnetic beads prepared in Example 1 and 500 μL of the sample to be tested into a 1.5ml centrifuge tube, shake at room temperature (18-27°C) for 50-60 minutes;

[0062] 2) Place the centrifuge tube on the magnet for 5 minutes to completely separate the magnetic beads from the solution, and use a pipette to remove the sample matrix from the bottom of the centrifuge tube to obtain the immunomagnetic beads-Shewanella Oneida immune complex;

[0063] 3) Wash the complex obtained in 2) three times with PBS washing solution containing 4-6% (v / v) Tween 20, wash once with pure water, and transfer to a 96-well plate. Add 100 μL of chromogenic solution (0.1-5 mM hydrogen peroxide, 90-110 mM sodium citrate, 190-210 mM disodium hydrogen phosphate, 0.3-0.35 mM tetramethylbenzidine, pH 4-5), room temperature (18-27 ℃) color reaction 5 ~ 60min;

[0064] 4) Add 50 μL of 2M sulfuric acid solu...

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Abstract

The invention discloses immunomagnetic bead and a method for rapidly detecting Shewanella oneidensis. A preparation method of the immunomagnetic bead is characterized in that the immunomagnetic bead can be obtained by incubating a Shewanella oneidensis specific antibody and activated carboxylated polystyrene immunomagnetic microsphere. The rapid detection method comprises the following steps: placing the immunomagnetic bead in a sample solution, fully combining the antibody of the immunomagnetic bead and the Shewanella oneidensis somatic antigen; removing a sample matrix to obtain an immunomagnetic bead-Shewanella oneidensis immune complex, washing, adding a coloured solution to perform a color development reaction, adding a sulfuric acid solution for stopping the color development reaction, detecting the OD450nm value, if the OD450nm value is more than 0.029, the sample contains Shewanella oneidensis. The method has the advantages that the sensitivity is high, the detection limit is 5.0*103cfu / ml, the operation is convenient and rapid, the method can be used for on-site detection; the specificity is good, and the detection result is nearly free from the influence of other common microbe.

Description

technical field [0001] The invention belongs to the field of microorganism detection, and relates to an immune magnetic bead and a method for rapidly detecting Shewanella Oneida. Background technique [0002] Shewanella oneida ( Shewanella oneidensis MR-1, S. oneidensis ) are Gram-negative facultative anaerobes belonging to the class Gamma-proteobacteria, widely found in seawater, marine sediments and freshwater environments. Under anaerobic conditions, Shewanella oneida can reduce heavy metal ions while degrading organic matter. It has broad application prospects in the field of environmental pollution remediation, for example, it can directly reduce water-soluble U (VI)) to U (IV). In addition, it can reduce thiosulfate to produce sulfur ions, which can then combine with heavy metal ions to form sulfide precipitates. In view of the application prospect of Shewanella oneida in the environmental field, it is of great significance to develop a low-cost rapid detection me...

Claims

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Application Information

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IPC IPC(8): G01N33/531G01N33/569G01N21/78
CPCG01N33/54326G01N33/56911G01N2446/80
Inventor 温俊林周顺桂陈俊华
Owner GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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