CN103293297A - Method for quickly separating salmonella - Google Patents
Method for quickly separating salmonella Download PDFInfo
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- CN103293297A CN103293297A CN2013102194187A CN201310219418A CN103293297A CN 103293297 A CN103293297 A CN 103293297A CN 2013102194187 A CN2013102194187 A CN 2013102194187A CN 201310219418 A CN201310219418 A CN 201310219418A CN 103293297 A CN103293297 A CN 103293297A
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Abstract
The invention discloses a method for separating foodborne pathogenic bacteria, and mainly relates to a method for separating salmonella. According to the method disclosed by the invention, basis is supplied to the better subsequent research on target bacteria. The method disclosed by the invention relates to the technical field of biology. The method comprises the following steps of: performing covalent coupling on dendritic polymers and antibodies, wrapping the dendritic polymers modified by the antibodies by long-chain biotin molecules, capturing the target bacteria in sample liquid by the dendritic polymers co-modified by the antibodies and the long-chain biotins, identifying nano magnetic beads modified by streptavidin, making the nano magnetic beads be coupled with the long-chain biotin dendritic polymers in the sample liquid, separating the captured bacteria, performing heavy suspension on the captured bacteria and the like. The captured target bacteria can be directly analyzed subsequently. Compared with the conventional bacterium magnetic separation method, the method disclosed by the invention is more suitable for magnetic separation of the bacteria in complicated substrates and the efficiency of separating the target bacteria in the sample is improved.
Description
Technical field
The present invention relates to biological technical field, specifically relate to the salmonella typhimurium separation method based on nanometer magnetic bead.
Background technology
Salmonella is the ubiquitous a kind of food-borne pathogens of occurring in nature, is accounted in food source, world pathogenic bacteria case by salmonellal food poisoning to hold pride of place.The salmonella infection mankind can cause diseases such as human acute diarrhea, vomiting, abdominal pain, high fever and septicemia.Salmonella typhimurium is one of common serotype of salmonella, according to 2001 in an investigation that relates to 29 countries in the whole world shows, salmonella typhimurium infects quantity and accounts for 17% of salmonella infection total amount then.Set up a kind of new method efficient, the fast detecting salmonella typhimurium and seem particularly urgent.In view of needs set up a kind of efficient, detection method fast, immune magnetic separation technique has obtained developing rapidly in the food-borne pathogens monitoring
The immunity magnetic separation technique is one of important component part of food-borne pathogens rapid screening technology, but this technology efficient capture, concentrates object bacteria in the enrichment liquid, improves the pathogenic bacteria detection sensitivity and shorten detection time.In recent years, based on the immunomagnetic separation (IMS) of magnetic micro-beads object bacteria antibody is connected on the magnetic bead, the magnetic bead that will be connected with antibody then drop in the sample liquid to object bacteria catch, enrichment, magnetic separates (concrete principle is seen Fig. 2 A).Yet should have many limitation based on the isolation technics of micron order immunomagnetic beads at present: 1) specific surface area of micron magnetic bead is less relatively, has reduced magnetic capture efficient; 2) because the particle properties of micron magnetic bead self, by heterogeneous reaction (multiphase reaction) combination, need the longer time go specificity to catch bacterial cell in the food substrate usually between itself and the bacterial cell; 3) micron magnetic bead monodispersity is relatively poor, and precipitation takes place self to assemble or form in food substrate liquid easily; 4) traditional immune magnetic separation technique, directly be coupled to antibody molecule on the immunomagnetic beads often, this process usually can cause the activity of antibody to reduce widely and cause the direction in space of antibody to change having increased space steric effect between antibody, thereby having reduced the capture rate 5 of antibody) food substrate character is complicated and assorted bacteria concentrations wherein non-purpose pathogenic bacteria are big, the micron magnetic bead is easy to generate non-specific adsorption, is difficult to realize the specific isolation to purpose bacterium in the food sample liquid; 6) excessive concentration of micron magnetic bead can cause the breakage (magnetic field causes the cell surface magnetic bead to be attracted each other, cell is squeezed even break) of bacterial cell, causes the failure that separates.7) during magnetic bead coupling antibody, generally adopt hydrophobic absorption or chemical coupling mode will have active antibody and be connected in magnetic bead surfaces.Antibody and magnetic bead surfaces distance are too near, and the hydrophobic or strong hydrophilicity group of the character of magnetic bead own and remained on surface thereof causes that easily the antibody space conformation changes, and cause the antibody biologically active to descend.
Summary of the invention
At the defective of prior art, the purpose of this invention is to provide a kind of capture rate height, easy, disengaging time weak point, the method for (less than 30 T/m) quick from complex matrices, special separation purpose bacterium salmonella typhimurium under the low gradient magnetic.
The fast separating process of salmonella typhimurium, may further comprise the steps: (1) takes by weighing 1 mg dendritic, be suspended in 4 mL, 0.01 mol/L, in the pH 8.0 PBS phosphate buffers, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration that makes glutaraldehyde is 3%, room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; Drip 3.5 mg salmonella typhimurium specific antibodies, room temperature reaction 24 h under the rotating speed of shaking table 150 r/min; Decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution freeze drying that obtains is got dendritic-antibody complex; (2) get 15 mg long-chain biotins, be suspended in 4 mL 0.01mol/L pH, the 8.0 PBS phosphate buffers, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration that makes glutaraldehyde is 3%, room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; Add 1.59 mg dendritic-antibody complexes, room temperature reaction 24 h under the rotating speed of shaking table 150 r/min; Decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution freeze drying that obtains is got long-chain biotin-dendritic-antibody complex; (3) get 1 mL testing sample solution, adding 0.1 mg salmonella typhimurium antibody and the co-modified dendritic of long-chain biotin is step (2) long-chain biotin-dendritic-antibody complex, place on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm; Add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the mixing instrument, with the rotating speed of 30 rpm incubated at room 15 min again, insert conventional magnetic force frame and separate 3 min; (4) after the magnetic separation, after deionized water cleans gently, mix the resuspended nanometer magnetic bead-Streptavidin-long-chain biotin-dendritic-antibody-salmonella typhimurium antigenic compound that is enriched with salmonella typhimurium that namely gets with the PBS damping fluid.
Described dendritic is amidized polyamide-amide type dendritic, and its molecular weight is 42000 Da.Structure such as Fig. 1.
Described nanometer magnetic bead particle diameter is 20-50 nm, is preferably 30 nm.
Dendritic is realized the covalent coupling of dendritic and antibody by the carboxyl of amino and salmonella typhimurium specific antibody.
Dendritic is realized the covalent coupling of dendritic and long-chain biotin by the carboxyl of amino and long-chain biotin molecule; Add excessive long-chain biotin to guarantee exposed amino sites on the sealing dendritic.
Concrete principle is seen Fig. 2 B.
This method is applicable to the separation purpose bacterium from sample substrate, and particularly suitable separates the purpose bacterium from complex matrices.Food samples comprises the food material after all kinds of fresh or freezing processing, as products such as fresh vegetables, meat, seafood and milks.Sample preparation gets final product according to conventional treatment method, makes solution to be measured after sample is pulverized.
Adopt technical solution of the present invention to have following beneficial effect:
1, the present invention is by the cascade enlarge-effect of dendritic, magnetic bacterium signal exponentially level is enlarged, under lower magnetic field intensity, just can realize the separation of magnetic bacterium, and in the identical time, compare than routine immunization magnetic bead separation method, it is stronger to be separated to purpose bacterium ability, is specially adapted to the separation of complex sample, as food samples, whole blood sample etc.At slow, the demanding defective in magnetic field of purpose bacterium speed in the 20-50 nm immunomagnetic beads separate complex matrix sample behind the simple employing antibody modification, adopt dendritic to realize the amplification of nanometer magnetic bead magnetic signal, thereby improved purpose bacterium separation efficiency in the complex matrices sample, realized that purpose bacterium specificity is separated fast in (less than 30 T/m) are complicated under low gradient magnetic the food substrate.
2, this programme has been avoided in the conventional method antibody molecule being coupled to magnetic bead surfaces and has been caused antibody activity to reduce and sterically hindered big shortcoming for antibody molecule is coupled on the dendritic.
3, the present invention adopts dendritic, can make reaction solution more stable, and difficult the precipitation increased the chance that antibody molecule contacts with object bacteria, is conducive to improve capture rate; Simultaneously, be connected with a large amount of long-chain biotin molecules on the dendritic, the nanometer magnetic bead that can modify in conjunction with Streptavidin, thus make on the dendritic in conjunction with a large amount of nanometer magnetic beads, realize the cascade amplification of magnetic bacterium signal, be conducive to shorten the disengaging time of magnetic bacterium.
4, with behind nanometer magnetic bead (20-50 nm) the replacement micron order magnetic particle, because the nanometer magnetic bead particle diameter is little, specific surface area is big, the steric hindrance of being combined with bacterium surface antigen is little, the covering efficient of bacterium surface magnetic bead significantly improves, and the bacterium of magnetic nano particle subcovering can keep normal shape, and nanometer magnetic bead also has dispersed and stable preferably in complex matrices, so the use of nanometer magnetic bead can overcome above-mentioned all because the defective of using the micron magnetic bead to cause.
5, the present invention is in detachment process, introduced dendritic, be connected with a large amount of long-chain biotin molecules on the dendritic, can be special and high-affinity ground be dispersed in that coupling has the identification of Streptavidin nanometer magnetic bead in the matrix solution, thereby make on the dendritic in conjunction with a large amount of nanometer magnetic beads, increase the magnetic bead quantity of target bacteria surface combination greatly, realized under magnetic field, separating fast the target bacteria of catching.Comparing with traditional bacterial magnetic separation method, is stabilized nano magnetic bead more in matrix because of what add, and this method is applicable to that more in complex matrices bacterium being carried out magnetic separates, and has improved purpose bacterium separation efficiency in the complex matrices sample.
6, during magnetic bead coupling antibody, generally adopt hydrophobic absorption or chemical coupling mode will have active antibody and be connected in magnetic bead surfaces.Antibody and magnetic bead surfaces distance are too near, and the hydrophobic or strong hydrophilicity group of the character of magnetic bead own and remained on surface thereof causes that easily the antibody space conformation changes, and cause the antibody biologically active to descend.Yet this experimental program is introduced dendritic in coupling process, and it has certain space size, thereby makes antibody molecule away from magnetic bead and magnetic bead surfaces, has avoided the adverse effect of the character of magnetic bead own and surperficial antagonist molecule.Simultaneously, the dendritic of introducing but can not influence the antibody space conformation, thereby has played the bioactive effect of protection antibody molecule.
Description of drawings
The end modified structural representation that amino dendritic is arranged of Fig. 1.
The operational flowchart of the conventional magnetic separation technique (A) of Fig. 2 and magnetic separation technique (B) involved in the present invention.
Embodiment
In order to make the present invention clearer, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explaining the present invention, and be not used in restriction the present invention.
The long-chain biotin is for buying in the carboxylated long-chain biotin of U.S. Thermo Fisher Scientific company (EZ-Link Sulfo-NHS-LC-Biotin, molecular weight 556.59).
The nanometer magnetic bead (30 nm) that is modified with Streptavidin is bought the Ocean NanoTech company in the U.S..
Dendritic is amidized polyamide-amide type dendritic, and its molecular weight is 42000 Da, available from Weihai Chen Yuan new chemical materials company limited.
Conventional magnetic force frame separates magnetic field intensity less than 30T/m.
N-maloyl imines NHSS, ethyl 3-(3-dimethylamino) carbodiimide hydrochloride EDC etc. is conventional reagent, repeats no more.
0.1%PBST compound method: 8.0 g NaCl, 0.2 g KCl, 0.24 g KH
2PO
4, 1.44 g Na
2HPO
4Be dissolved in the 800 mL distilled water, adjust pH to 7.4 with 5 M NaOH, be settled to 1000 mL again and namely get 0.01 M PBS.Volume ratio with 1/1000 (V/V) adds Tween 20 again, namely obtains 0.1%PBST.
Embodiment 1
1. dendritic-antibody complex prepares according to following steps:
(1) takes by weighing the amidized polyamide-amide type of 1 mg dendritic dendritic, be suspended in the 4 mL phosphate buffers (pH 8.0 for PBS, 0.01 mol/L), the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration that makes glutaraldehyde is 3%.Room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min;
(2) add 3.5mg salmonella typhimurium specific antibody to above-mentioned drips of solution, room temperature reaction 24 h under the rotating speed of shaking table 150 r/min;
(3) above-mentioned solution decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution freeze drying that obtains.
2. long-chain biotin-dendritic-antibody complex prepares according to following steps:
((1) whenever gets 15 mg long-chain biotins, be suspended in the 4 mL phosphate buffers (pH 8.0 for PBS, 0.01 mol/L), and the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration that makes glutaraldehyde is 3%.Room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min;
(2) 1.59 mg dendritic-antibody complexes are joined in the above-mentioned solution room temperature reaction 24 h under the rotating speed of shaking table 150 r/min;
(3) above-mentioned solution decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution freeze drying that obtains.。
3. enrichment is caught: get testing sample solution 1 mL, add 0.1 mg long-chain biotin-dendritic-antibody complex, place on the mixing instrument, with rotating speed incubated at room 15 min formation long-chain biotin-dendritic-antibody-salmonella typhimurium antigenic compound of 30 rpm; Add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the mixing instrument, with the rotating speed of 30 rpm incubated at room 15 min again, centrifuge tube is inserted conventional magnetic force frame separate 3 min;
4. after deionized water cleaned gently, mixing the resuspended compound that is enriched with the salmonella typhimurium salmonella typhimurium that namely gets with the PBS damping fluid was nanometer magnetic bead-Streptavidin-biotin-dendritic-antibody-salmonella typhimurium antigen.
The experiment of embodiment 2 concentration effects
(1) getting 1 mL concentration is 10
4The salmonella typhimurium of cfu/mL is in the aseptic centrifuge tube of 1.5 mL, and centrifugal 5 min of 12000 rpm abandon supernatant, and is resuspended with the aseptic PBS solution of equal-volume.
(2) enrichment is caught: technical solution of the present invention group (the dendritic group that salmonella typhimurium antibody and long-chain biotin are co-modified), the nanometer magnetic bead group that the salmonella typhimurium specific antibody is modified, the micron magnetic bead group enrichment purpose bacterium that the salmonella typhimurium specific antibody is modified are set respectively.
(3) after magnetic separates, supernatant is poured in the aseptic centrifuge tube, separated the immunomagnetic beads of catching salmonella typhimurium and then clean twice with PBST, mix, and with the resuspended immunomagnetic beads compound of the aseptic PBS solution of 1 mL.
(4) capture rate calculates: after the resuspended liquid of purpose bacterium of each group enrichment is carried out gradient dilution, to each gradient counting, calculate the capture rate of object bacteria by the capture rate formula with dull and stereotyped, test triplicate at every turn.Each computing formula of organizing capture rate is as follows: (total plate count/all totals number of bacteria of being adsorbed by enrichment) * 100%.
Describedly respectively organize enrichment to catch the scheme of purpose bacterium as follows:
A. purpose bacterium scheme such as embodiment 1 are caught in technical solution of the present invention group (the dendritic group that salmonella typhimurium antibody and long-chain biotin are co-modified) enrichment, and be specific as follows:
Be that biotin-dendritic-antibody complex joins and contains in the object bacteria centrifuge tube with 0.1 mg salmonella typhimurium antibody and the co-modified dendritic of biotin, place on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm.Add 0.1 mg then and be modified with the nanometer magnetic bead of Streptavidin, place on the mixing instrument, with the rotating speed of 30 rpm incubated at room 15 min again.At last, centrifuge tube is inserted conventional magnetic force frame and separate 3 min.
B. it is specific as follows that purpose bacterium scheme is caught in the nanometer magnetic bead group enrichment of salmonella typhimurium specific antibody modification:
The nanometer magnetic bead that the salmonella typhimurium specific antibody that 0.1 mg is prepared is modified joins and contains in the object bacteria centrifuge tube, places on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm.At last, centrifuge tube is inserted conventional magnetic force frame and separate 3 min.
The nanometer magnetic bead preparation that described salmonella typhimurium specific antibody is modified: (1) is got 10 mg nanometer magnetic beads (30 nm do not have the coupling Streptavidin) and is used absolute ethyl alcohol, 1 M NaOH successively, each washing of 1 M HCl once, PBS(0.02 M, pH 4.0) give a baby a bath on the third day after its birth time, aseptic PBS is resuspended.Add NHSS 0.4 mg, EDC 0.35 mg places to keep magnetic bead to suspend on the mixing instrument 37 ℃ of activation 2 h.(2) the magnetic force frame reclaims magnetic bead, and PBS(0.02 M, pH 4.0) after the washing three times, magnetic bead is resuspended among the aseptic PBS, adds 80 μ g salmonella typhimurium specific antibodies by every mg magnetic bead, places 37 ℃ of coupling 2 h on the mixing instrument.(3) add the monoethanolamine room temperature and seal 2 h.Magnet stand reclaims magnetic bead, PBS washing three times, and 10 ml PBS(contain 0.05% NaN
3, 0.5% BSA, pH 7.4) resuspended immunomagnetic beads and standby in 4 ℃ of refrigerators preservations.
C. it is specific as follows that purpose bacterium scheme is caught in the micron magnetic bead group enrichment of salmonella typhimurium specific antibody modification:
The micron magnetic bead that the salmonella typhimurium specific antibody that 0.1 mg is prepared is modified joins and contains in the object bacteria centrifuge tube, places on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm.At last, centrifuge tube is inserted conventional magnetic force frame and separate 3 min.
The micron magnetic bead preparation that described salmonella typhimurium specific antibody is modified: (1) gets 10 mg micron magnetic bead (1150 nm, do not have the coupling Streptavidin) use absolute ethyl alcohol successively, 1 M NaOH, each washing of 1 M HCl once, PBS(0.02 M, pH 4.0) give a baby a bath on the third day after its birth time, aseptic PBS is resuspended.Add NHSS 0.4 mg, EDC 0.35 mg places to keep magnetic bead to suspend on the mixing instrument 37 ℃ of activation 2 h.(2) the magnetic force frame reclaims magnetic bead, and PBS(0.02 M, pH 4.0) after the washing three times, magnetic bead is resuspended among the aseptic PBS, adds 80 μ g salmonella typhimurium specific antibodies by every mg magnetic bead, places 37 ℃ of coupling 2 h on the mixing instrument.(3) add the monoethanolamine room temperature and seal 2 h.Magnet stand reclaims magnetic bead, PBS washing three times, and 10 ml PBS(contain 0.05% NaN
3, 0.5% BSA, pH 7.4) resuspended immunomagnetic beads and standby in 4 ℃ of refrigerators preservations.
It is as follows that each organizes capture rate:
| The micron magnetic bead group capture rate that the salmonella typhimurium specific antibody is modified | The nanometer magnetic bead group capture rate that the salmonella typhimurium specific antibody is modified | The dendritic group capture rate that salmonella typhimurium antibody and long-chain biotin are co-modified |
| 62.2% | 25.6% | 93.5% |
Experimental result shows, the capture rate of the micron magnetic bead group that the salmonella typhimurium specific antibody is modified is apparently higher than the capture rate of nanometer magnetic bead group, this explanation contrast nanometer magnetic bead group, because micron magnetic bead volume is big, magnetic is strong, at short notice just can the more object bacteria of separation and concentration.But, the capture rate of technical solution of the present invention group is far longer than the micron magnetic bead group that the salmonella typhimurium specific antibody is modified again, this shows that technical solution of the present invention can increase object bacteria nano surface magnetic bead coverage rate by dendritic, thereby magnetic is improved greatly, and then realized (3min) efficient separation and concentration salmonella typhimurium at short notice.
Experiment is caught in embodiment 3 enrichments
Conventional magnetic force frame disengaging time is 30min, and all the other are with embodiment 2.
It is as follows that each organizes capture rate:
| The micron magnetic bead group capture rate that the salmonella typhimurium specific antibody is modified | The nanometer magnetic bead group capture rate that the salmonella typhimurium specific antibody is modified | The dendritic group capture rate that salmonella typhimurium antibody and long-chain biotin are co-modified |
| 63.5% | 42.6% | 94.1% |
Experimental result shows, separate 3min among the comparative example 2, when disengaging time reaches 30min, three groups capture rate all is improved, particularly the capture rate of the nanometer magnetic bead group of salmonella typhimurium specific antibody modification improves the most obvious, this shows the capture rate that can improve the nanometer magnetic bead group by time expand widely, but it still is lower than the capture rate of short time co-modified dendritic group of salmonella typhimurium antibody and long-chain biotin when separating (3min).This shows technical solution of the present invention (3min) efficient separation and concentration salmonella typhimurium at short notice.
Embodiment 4
Aseptic meat is pulverized, made testing sample solution in the usual way, add salmonella typhimurium and regulate bacterium colony concentration to 10
4Cfu/mL is standby.
The salmonella typhimurium antibody and the co-modified dendrimer (0.1 mg) of long-chain biotin that prepare are joined respectively in the sample solution, place on the mixing instrument, with rotating speed incubated at room 15 min＜i TranNum=" 158 " of 30 rpm 〉.</i>Add the nanometer magnetic bead (0.1 mg) be modified with Streptavidin then, place on the mixing instrument, with the rotating speed of 30 rpm incubated at room 15 min again.At last, conventional magnetic force frame separates 3 min.After magnetic separates, supernatant is poured in the aseptic centrifuge tube, separated the immunomagnetic beads of catching salmonella typhimurium and then clean twice with PBST, mix, and with the resuspended immunomagnetic beads of the aseptic PBS solution of 1 mL.Capture rate such as embodiment 2 methods obtain, and all the other are with embodiment 2.The results are shown in Table 1, show the salmonella typhimurium in this programme energy efficiently concentrating sample separation.
Embodiment 5
Germ-free milk is the sample testing sample solution, adds salmonella typhimurium and regulates bacterium colony concentration to 10
4Cfu/mL.All the other are with embodiment 4.
Embodiment 6
Aseptic grates vegetables is made testing sample solution in the usual way, adds salmonella typhimurium and regulates bacterium colony concentration to 10
4Cfu/mL.
Embodiment 7
Testing sample is aseptic whole blood, adds salmonella typhimurium and regulates bacterium colony concentration to 10
4Cfu/mL.All the other are with embodiment 4.
The comparison of salmonella typhimurium separating effect in the different actual samples of table 1
| Actual sample | The dendritic group capture rate that salmonella typhimurium antibody and long-chain biotin are co-modified |
| Embodiment 2 meats | 81.5% |
| Embodiment 3 milk | 82.5% |
| Embodiment 4 vegetables | 84.3% |
| Embodiment 5 whole bloods | 80.5% |
The above only is preferred embodiment of the present invention, not in order to limiting the present invention, all any modifications of doing within the spirit and principles in the present invention, is equal to and replaces and improvement etc., all should be included within protection scope of the present invention.
Claims (3)
1. the fast separating process of salmonella typhimurium, it is characterized in that may further comprise the steps: (1) takes by weighing 1 mg dendritic, be suspended in 4 mL, 0.01 mol/L, in the pH 8.0PBS phosphate buffer, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration that makes glutaraldehyde is 3%, room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; Drip 3.5 mg salmonella typhimurium specific antibodies, room temperature reaction 24 h under the rotating speed of shaking table 150 r/min; Decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution freeze drying that obtains is got dendritic-antibody complex; (2) get 15 mg long-chain biotins, be suspended in 4 mL, 0.01 mol/L pH, the 8.0 PBS phosphate buffers, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration that makes glutaraldehyde is 3%, room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; Add 1.59 mg dendritic-antibody complexes, room temperature reaction 24 h under the rotating speed of shaking table 150 r/min; Decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution freeze drying that obtains is got long-chain biotin-dendritic-antibody complex; (3) get 1 mL testing sample solution, adding 0.1 mg salmonella typhimurium antibody and the co-modified dendritic of long-chain biotin is step (2) long-chain biotin-dendritic-antibody complex, place on the mixing instrument, with rotating speed incubated at room 15 min of 30 rpm; Add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the mixing instrument, with the rotating speed of 30 rpm incubated at room 15 min again, insert conventional magnetic force frame and separate 3 min; (4) after the magnetic separation, after deionized water cleans gently, mix the resuspended nanometer magnetic bead-Streptavidin-long-chain biotin-dendritic-antibody-salmonella typhimurium antigenic compound that is enriched with salmonella typhimurium that namely gets with the PBS damping fluid.
2. method according to claim 1 is characterized in that described dendritic is amidized polyamide-amide type dendritic, and its molecular weight is 42000 Da.
3. method according to claim 1 is characterized in that described nanometer magnetic bead particle diameter is 20-50 nm, is preferably 30 nm.
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| CN109929813A (en) * | 2019-03-04 | 2019-06-25 | 华中农业大学 | Salmonella bacteriophage nanometer magnetic bead conjugate and its concentration and separation kit |
| CN112505117A (en) * | 2020-11-11 | 2021-03-16 | 军事科学院军事医学研究院军事兽医研究所 | Ferrocene nanoflower, electrochemical aptamer biosensor system and preparation method and application thereof |
| CN113801855A (en) * | 2021-08-25 | 2021-12-17 | 华中农业大学 | Salmonella typhimurium bacteriophage T102 and application thereof in enrichment and separation of salmonella |
| CN113801855B (en) * | 2021-08-25 | 2023-08-25 | 华中农业大学 | Salmonella typhimurium phage T102 and application thereof in enrichment and separation of salmonella |
| CN114369551A (en) * | 2022-01-07 | 2022-04-19 | 中国农业科学院油料作物研究所 | Preparation method and application of magnetic nanoparticles for rapidly separating bifidobacterium bifidum |
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