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Efficient method for separating meticillin-resistant Sta-phylococcusaureus

A methicillin-resistant and staphylococcus-resistant technology, applied in the field of isolation of pathogenic bacteria based on nano-magnetic beads, can solve the problems of separation failure, high concentration of miscellaneous bacteria, poor monodispersity of micron magnetic beads, etc., to increase the chance of contact, Effects of improving separation efficiency and shortening separation time

Inactive Publication Date: 2015-04-15
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current separation technology based on micron-scale immunomagnetic beads has many limitations: 1) The specific surface area of ​​micron-sized magnetic beads is relatively small, which reduces the capture efficiency of magnetic beads; Bacterial cells are combined through a multiphase reaction (multiphase reaction), which usually takes longer to specifically capture bacterial cells in the matrix; 3) Micron magnetic beads have poor monodispersity and are prone to self-aggregation or Precipitation; 4) The traditional immunomagnetic separation technology often directly couples antibody molecules to immunomagnetic beads. This process often leads to a greatly reduced activity of the antibody and changes in the spatial direction of the antibody, increasing the inter-antibody interaction. steric hindrance effect, which reduces the capture efficiency of antibodies. 5) Whole blood, secretions and other matrices have complex properties and the concentration of non-target pathogenic bacteria is high. Micron magnetic beads are prone to non-specific adsorption, and it is difficult to achieve sample detection. Specific separation of target bacteria in liquid; 6) Too high concentration of micron magnetic beads will cause damage to bacterial cells (magnetic field causes magnetic beads on the surface of cells to attract each other, causing cells to be squeezed or even ruptured, resulting in failure of separation; 7) Magnetic When beads are coupled to antibodies, hydrophobic adsorption or chemical coupling is generally used to couple active antibodies to the surface of magnetic beads.
The distance between the antibody and the surface of the magnetic bead is too close, the nature of the magnetic bead itself and the residual hydrophobic or strong hydrophilic groups on the surface are likely to cause changes in the spatial conformation of the antibody, resulting in a decrease in the biological activity of the antibody

Method used

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  • Efficient method for separating meticillin-resistant Sta-phylococcusaureus
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1. The broom molecule-antibody complex is prepared according to the following steps:

[0030] (1) Weigh 1.0 mg of aminated broom molecules, suspend in 4 mL of phosphate buffer (PBS, 0.01 mol / L, pH 8.0), stir and add 545 μL of 25% glutaraldehyde aqueous solution dropwise to make glutaraldehyde The final concentration was 3%. React at room temperature for 3.5 h at a rotating speed of 150 r / min on a shaker;

[0031] (2) Add methicillin-resistant Staphylococcus aureus dropwise to the above solution MRSA Add 1 mL of specific antibody to make the final concentration reach about 3 mg / mL. React at room temperature for 24 hours at a shaking table of 150 r / min;

[0032] (3) The above solution was spin-dried under reduced pressure, dissolved in deionized water, and dialyzed in PBS and deionized water for 1 day; after the dialysis, the obtained solution was freeze-dried.

[0033] 2. The long-chain biotin-broom molecule-antibody complex is prepared according to the f...

Embodiment 2

[0039] Example 2 Enrichment effect experiment

[0040] (1) Take 1 mL of concentration as 10 4 cfu / mL MRSA Centrifuge at 12,000 rpm for 5 min in a 1.5 mL sterile centrifuge tube, discard the supernatant, and resuspend with an equal volume of sterile PBS solution.

[0041] (2) Enrichment and capture: respectively set the technical solution group of the present invention ( MRSA Antibody and long-chain biotin co-modified broom group), MRSA Specific antibody-modified nano-magnetic bead set, MRSA Specific antibody-modified micron magnetic bead group enriches target bacteria.

[0042] (3) After magnetic separation, pour the supernatant into a sterile centrifuge tube, and capture the MRSA The immunomagnetic beads were washed twice with PBST, mixed well, and the immunomagnetic bead complex was resuspended with 1 mL sterile PBS solution.

[0043] (4) Capture rate calculation: After gradient dilution of the enriched target bacteria resuspension in each group, count each gradi...

Embodiment 3

[0056] Example 3 Enrichment capture experiment

[0057] Conventional magnetic stand separation time is 30min, and all the other are with embodiment 2.

[0058] The catch rate of each group is as follows:

[0059] MRSA Capture efficiency of specific antibody-modified micron magnetic bead sets MRSA Capture efficiency of specific antibody-modified nanomagnetic bead sets MRSA Capture efficiency of broom groups co-modified with antibodies and long-chain biotin 56.7% 38.9% 91.2%

[0060] Experimental result shows, separates 3min among the comparative example 2, when separation time reaches 30min, the capture efficiency of three groups has all been improved, especially MRSA The capture efficiency of the specific antibody-modified nano-magnetic bead group is the most obvious, which shows that the capture efficiency of the nano-magnetic bead group can be greatly improved by extending the time, but it is still lower than the short-time separation (3mi...

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Abstract

The invention discloses a method of enrichment separation of meticillin-resistant Sta-phylococcusaureus (MRSA), provides a better basic for follow-up study of target bacteria, and relates to the field of biology technology. The method comprises steps of: covalently coupling broom molecules and antibodies, coating antibody-modified broom molecules with long-chain biotin molecules, capturing target bacteria in a sample solution via broom molecules that are modified by the antibodies and the long-chain biotin molecules, identifying streptavidin-modified nano-magnetic-beads and coupling with long-chain biotinylated broom molecules in the sample solution, separating and re-suspending the captured bacteria. Because the re-suspending solution can be used for follow-up analyses directly, the method provide by the invention is even more suitable for magnetic separation of bacteria in complex matrixes compared with conventional magnetic separation of bacteria, and rises the separating efficiency of target bacteria in samples.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for isolating pathogenic bacteria based on nano magnetic beads. Background technique [0002] Methicillin-resistant Staphylococcus aureus ( Meticillin-resistant Staphylococcus aureus, MRSA ) is the most common pathogen of nosocomial infections. It is also an important pathogen causing suppurative diseases, food poisoning and toxic shock syndrome, and the infection caused by it has become a rather difficult problem in clinical treatment. Its purulent infection can range from small skin lesions such as boils and carbuncles to serious infections such as pneumonia, mastitis, phlebitis, meningitis, and inflammation of the urinary system, and can reach deep to cause osteomyelitis and endocarditis. MRSA It is a serious pathogenic bacterium of nosocomial infection, which is often found around the indwelling catheter in the body after trauma or operation. MRSA It can also cause fo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N13/00C12N1/20C12R1/445
Inventor 许恒毅熊勇华黄小林郭亮江湖
Owner NANCHANG UNIV
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