Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for separating staphylococcus aureus (SA)

A staphylococcus, golden yellow technology, applied in the biological field, can solve the problems of separation failure, poor monodispersity of micron magnetic beads, small specific surface area, etc., and achieve the effect of increasing contact opportunities, shortening separation time and improving separation efficiency

Inactive Publication Date: 2015-03-11
NANCHANG UNIV
View PDF1 Cites 0 Cited by
  • 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) Compared with nano-scale magnetic beads, the specific surface area of ​​micron-scale magnetic beads is small, which reduces the capture efficiency of magnetic beads; Particle nature, which combines with bacterial cells through a multiphase reaction (multiphase reaction), usually takes longer to specifically capture bacterial cells in the food matrix; 3) Micron magnetic beads have poor monodispersity 4) The traditional immunomagnetic separation technology often couples antibody molecules directly to immunomagnetic beads, which often leads to a large decrease in antibody activity and a change in the spatial direction of the antibody, increasing the The steric hindrance effect between antibodies is increased, thereby reducing the capture efficiency of antibodies; 5) The nature of the food matrix is ​​complex and the concentration of non-target pathogenic bacteria (miscellaneous bacteria) is large, and micron magnetic beads are prone to non-specific adsorption, making it difficult to achieve Specific separation of target bacteria in food sample liquid; 6) Excessive concentration of micron magnetic beads will cause damage to bacterial cells (the magnetic field causes the magnetic beads on the cell surface to attract each other, causing the cells to be squeezed or even ruptured), resulting in failure of separation; (7) When magnetic beads are coupled to antibodies, hydrophobic adsorption or chemical coupling is generally used to link active antibodies to the surface of magnetic beads
If the distance between the antibody and the surface of the magnetic bead is too close, due to the nature of the magnetic bead itself and the hydrophobic or strong hydrophilic groups remaining on the surface, it is easy to cause changes in the spatial conformation of the antibody, resulting in a decrease in the biological activity of the antibody

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for separating staphylococcus aureus (SA)
  • Method for separating staphylococcus aureus (SA)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. The dendrimer-antibody complex is prepared according to the following steps:

[0031] (1) Weigh 1.0 mg of aminated dendrimers, 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 of aldehyde was 3%. React at room temperature for 3.5 h at a rotating speed of 150 r / min on a shaker;

[0032] (2) Add Staphylococcus aureus dropwise to the above solution SA Specific antibody 1 mL, so that the final concentration reached about 3 mg / mL. React at room temperature for 24 h at the speed of the shaker at 150 r / min;

[0033] (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.

[0034] 2. The long-chain biotin-dendrimer-antibody complex is prepared according to the following steps:

[0...

Embodiment 2

[0040] Example 2 Enrichment effect experiment

[0041] (1) Take 1 mL of concentration as 10 4 cfu / mL SA 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.

[0042] (2) Enrichment and capture: respectively set the technical solution group of the present invention ( SA dendrimers co-modified with antibodies and long-chain biotin), SA Specific antibody-modified nano-magnetic bead set, SA Specific antibody-modified micron magnetic bead group enriches target bacteria.

[0043] (3) After magnetic separation, pour the supernatant into a sterile centrifuge tube, and capture the SA The immunomagnetic beads were washed twice with PBST, mixed well, and the immunomagnetic bead complex was resuspended with 1 mL sterile PBS solution.

[0044] (4) Capture rate calculation: After gradient dilution of the enriched target bacteria resuspension in each group, count each gradient ...

Embodiment 3

[0057] Example 3 Enrichment capture experiment

[0058] Conventional magnetic stand separation time is 30 min, all the other are with embodiment 2.

[0059] The catch rate of each group is as follows:

[0060] SA Capture efficiency of specific antibody-modified micron magnetic bead sets SA Capture efficiency of specific antibody-modified nanomagnetic bead sets SA Capture efficiency of dendrimers co-modified with antibodies and long-chain biotin 60.71% 38.1% 91.8%

[0061] Experimental result shows, separates 3 min among the comparative example 2, and when separation time reaches 30 min, the capture efficiency of three groups has all been improved, especially SA 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 (3 min) SA C...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
particle diameteraaaaaaaaaa
particle diameteraaaaaaaaaa
Login to View More

Abstract

The invention discloses a method for enriching and separating staphylococcus aureus (SA), provides a good basis to the subsequent research on a target bacterium, and relates to the technical field of biology. The method comprises the following steps: performing covalent coupling on a dendrimer and an antibody; coating the antibody modified dendrimer with a long-chain biotin molecule; capturing a target bacterium in a sample liquid by the antibody and long-chain biotin comodified dendrimer; identifying and coupling the long-chain biotin dendrimer in the sample liquid by using streptavidin modified nano-magnetic beads; and carrying out separation and weight suspending on the captured bacterium, wherein the weight suspending solution can be directly used for subsequent analysis. Compared with a traditional bacterium magnetic separation method, the method is more suitable for magnetically separating bacterium in a complex matrix, so that the target bacterium separation efficiency in a sample can be improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for isolating food-borne pathogenic bacteria based on nano magnetic beads. Background technique [0002] Foodborne pathogen contamination is one of the major problems of food safety in my country. According to WHO statistics, about one-third of people in developed countries are infected with food-borne diseases every year, and 2.2 million people in the world die every year due to food-borne diseases. In my country, the number of cases of food poisoning is between 200,000 and 400,000 per year, most of which are caused by food-borne pathogens except for accidents. Staphylococcus aureus (Staphylococcus aureus, SA) As one of the common food-borne pathogenic bacteria, it is the only pathogenic bacteria allowed to exist in a limited amount in quick-frozen brand food by the Ministry of Health of my country. However, the poisoning incidents caused by it occur from time to time, a...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12R1/445
Inventor 魏华许恒毅徐锋万翠香
Owner NANCHANG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products