CN103320421B - Efficient method for separating meticillin-resistant Sta-phylococcusaureus - Google Patents
Efficient method for separating meticillin-resistant Sta-phylococcusaureus Download PDFInfo
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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
The present invention relates to biomedical sector, specifically relate to the pathogenic bacterium separation method based on nanometer magnetic bead.
Background technology
Methicillin-resistant staphylococcus aureus (
meticillin-resistant Staphylococcus aureus, MRSA) be the former bacterium of most commonly encountered diseases of hospital infection.Also be the important pathogenic bacteria causing purulent disease, food poisoning and toxic shock syndrome, the infection caused by it to become in clinical treatment rather stubborn problem the sixth of the twelve Earthly Branches.Its pyogenic infection from little dermatosis as furuncle, carbuncle to serious infection as pneumonia, mazoitis, phlebitis, meningitis, Urinary system inflammation, and deep can be arrived cause osteomyelitis, endocarditis etc.
mRSAbe the serious pathogenic bacteria of one of hospital infection, be common in wound or Post operation, the surrounding catheter of intracorporeal indwelling.
mRSAalso causing food poisoning by discharging enterotoxin in food, in blood, discharging superantigen and cause toxic shock syndrome.
mRSAbe everlasting after Infective local or whole body causes nonspecific inflammation to react, and can cause unmanageable fatal septicemia.Found from 1961
mRSAsince, particularly after the eighties, this bacterium infection rate constantly rises, and resistance strengthens gradually.The ward infection caused by it and food poisoning happen occasionally, and therefore the technology pole of methicillin-resistant staphylococcus aureus is necessary fast, in efficiently concentrating sample separation in development.
Immunity magnetic separation technique is one of important component part of pathogenic bacterium rapid screening technology, and this technology can efficient capture, object bacteria in concentrated enrichment liquid, improves pathogenic microbes detect sensitivity.In recent years, object bacteria antibody is connected on magnetic bead by the immunomagnetic separation (IMS) based on magnetic micro-beads, then the magnetic bead being connected with antibody is dropped in sample liquid object bacteria is caught, enrichment, Magneto separate (concrete principle is shown in Fig. 2 A).But, many limitation should be there is based on the isolation technique of micron order immunomagnetic beads at present: 1) specific surface area of micron magnetic bead is relatively little, reduces magnetic capture efficiency; 2) due to the particle properties of micron magnetic bead self, combined by heterogeneous reaction (multiphase reaction) between itself and bacterial cell, usually need the time more grown to go specificity to catch bacterial cell in matrix; 3) micron magnetic bead monodispersity is poor, self assemble easily occurs in matrix liquid or forms precipitation; 4) traditional immune magnetic separation technique, often antibody molecule is directly coupled on immunomagnetic beads, this process usually can cause the activity of antibody to reduce widely and cause the direction in space of antibody to change the space steric effect increased between antibody, thus reduce the capture rate 5 of antibody) medium property such as whole blood, juice is complicated and wherein the miscellaneous bacteria concentration of non-object pathogenic bacterium is large, micron magnetic bead easily produces non-specific adsorption, is difficult to realize the specific isolation to object bacterium in sample liquid; 6) excessive concentration of micron magnetic bead can cause the breakage of bacterial cell (magnetic field causes cell surface magnetic bead to be attracted each other, and cell is squeezed and even breaks, cause be separated failure; 7), during magnetic bead coupled antibody, activated for tool antibody is connected in magnetic bead surfaces by general hydrophobic adsorbent or the chemical coupling mode of adopting.Too closely, the hydrophobic or strong hydrophilicity group of magnetic bead nature and remained on surface thereof easily causes antibody space conformation to change, and causes antibody bioactive to decline for antibody and magnetic bead surfaces distance.
Summary of the invention
For the defect of prior art, the object of this invention is to provide high, the easy disengaging time of a kind of capture rate short, the method for object bacterium methicillin-resistant staphylococcus aureus specificity sharp separation in the whole blood that (to be less than 30 T/m) under low gradient magnetic complicated, secretory product matrix.
Methicillin-resistant staphylococcus aureus fast separating process, comprise the following steps: (1) takes the broom molecule of 1.0 mg, be suspended in 4 mL 0.01 mol/L pH 8.0 PBS phosphoric acid buffers, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration of glutaraldehyde is made to be 3%, room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; Drip 1mL methicillin-resistant staphylococcus aureus
mRSAspecific antibody, makes its final concentration reach about 3 mg/mL, room temperature reaction 24 h under the rotating speed of shaking table 150 r/min; Decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize obtained to obtain broom molecule-antibody complex; (2) 15 mg long-chain biological elements are got, be suspended in 4 mL 0.01mol/L pH 8.0 PBS phosphoric acid buffers, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, makes the final concentration of glutaraldehyde be 3%, room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; Add 0.53 mg broom molecule-antibody complex, room temperature reaction 24 h under the rotating speed of shaking table 150 r/min; Decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize obtained to obtain long-chain biological element-broom molecule-antibody complex; (3) get 1 mL testing sample solution, add 0.15 mg
mRSAantibody and long-chain biological element co-modified broom molecule and step (2) long-chain biological element-broom molecule-antibody complex, be placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm; Add the nanometer magnetic bead that 0.15 mg is modified with Streptavidin, be placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm, conventional magnetic frame is separated 3 min; (4) after magneticseparation, after deionized water cleans gently, with PBS damping fluid mix resuspended namely obtain nanometer magnetic bead-Streptavidin-long-chain biological element-broom molecule-antibody of being enriched with methicillin-resistant staphylococcus aureus-
mRSAantigenic compound.
Described broom molecule is amidized linearizing polyamide-amide, and its molecular weight is 13000 Da.Structure is shown in Fig. 1.
Described nanometer magnetic bead particle diameter is 20-50 nm, is preferably 30 nm.
Broom molecule by amino and
mRSAthe carboxyl of specific antibody realizes the covalent coupling of broom molecule and antibody.
Broom molecule, by carboxyl that is amino and long-chain biological element molecule, realizes the covalent coupling of broom molecule and long-chain biological element; Add excessive long-chain biological element to ensure amino sites exposed on closed broom molecule.
Concrete principle is shown in Fig. 2 B.
Present method is specially adapted to the separation of complex sample, as food samples, whole blood sample, secretory product etc.Sample preparation is treatment process conveniently, as will be made solution to be measured after sample comminution.
Technical solution of the present invention is adopted to have following beneficial effect:
1, the present invention by means of the Cascaded amplification effect of broom molecule, magnetic bacterium signal exponentially level is expanded, the separation of magnetic bacterium just can be realized under lower magneticstrength, and within the identical time, comparatively routine immunization Beads enrichment method is compared, be separated to object bacterium ability stronger, be specially adapted to the separation of complex sample, as food samples, whole blood sample etc.For the defect that object bacterium speed is slow, magnetic field requirements is high in the simple 20-50 nm immuno magnetic cell separation complex matrices sample adopted after antibody modification, dendrimer is adopted to realize the amplification of nanometer magnetic bead magnetic signal, thus improve object bacterium separation efficiency in complex matrices sample, achieve object bacterium specificity sharp separation in the complex matrices that (is less than 30 T/m) under low gradient magnetic.
2, this programme is for be coupled on broom molecule by antibody molecule, avoid in ordinary method antibody molecule is coupled to magnetic bead surfaces cause antibody activity reduce and sterically hindered large shortcoming.
3, the present invention adopts broom molecule, reaction soln can be made more stable, not easily precipitate, add the chance that antibody molecule contacts with object bacteria, is conducive to improving capture rate; Simultaneously, broom molecule is connected with a large amount of long-chain biological element molecules, the nanometer magnetic bead can modified in conjunction with Streptavidin, thus makes on broom molecule in conjunction with a large amount of nanometer magnetic beads, achieve the Cascaded amplification of magnetic bacterium signal, be conducive to the disengaging time shortening magnetic bacterium.
4, after replacing micron order magnetic particle with nanometer magnetic bead (20-50 nm), because nanometer magnetic bead particle diameter is little, specific surface area is large, the steric hindrance be combined with bacterial surface antigen is little, the covering efficiency of bacterium surface magnetic bead significantly improves, and the bacterium of magnetic nano particle subcovering can keep normal shape, nanometer magnetic bead also has dispersed and stability preferably in complex matrices, and therefore the use of nanometer magnetic bead can overcome above-mentioned all defects owing to using micron magnetic bead to cause.
5, the present invention is in sepn process, introduce broom molecule, broom molecule is connected with a large amount of long-chain biological element molecules, can special and high-affinity ground be dispersed in coupling in matrix solution and have the identification of Streptavidin nanometer magnetic bead, thus make on broom molecule in conjunction with a large amount of nanometer magnetic beads, considerably increase the magnetic bead quantity of target bacteria surface bonding, achieve the target bacteria that sharp separation is caught under magnetic field.Compared with traditional bacterial magnetic separation method, be nanometer magnetic bead more stable in matrix because of what add, the method is more suitable for carries out Magneto separate to bacterium in complex matrices, improves object bacterium separation efficiency in complex matrices sample.
6, during magnetic bead coupled antibody, activated for tool antibody is connected in magnetic bead surfaces by general hydrophobic adsorbent or the chemical coupling mode of adopting.Too closely, the hydrophobic or strong hydrophilicity group of magnetic bead nature and remained on surface thereof easily causes antibody space conformation to change, and causes antibody bioactive to decline for antibody and magnetic bead surfaces distance.But this experimental program introduces broom molecule in coupling process, it has certain space size, thus makes antibody molecule away from magnetic bead and magnetic bead surfaces, avoids the disadvantageous effect of magnetic bead nature and surperficial antagonist molecule.Meanwhile, the broom molecule of introducing but can not affect antibody space conformation, thus serves the bioactive effect of protection antibody molecule.
Accompanying drawing explanation
Fig. 1 broom schematic arrangement.
The operational flowchart of the conventional magnetic separation technique (A) of Fig. 2 and magnetic separation technique involved in the present invention (B).
Embodiment
In order to make the present invention clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Long-chain biological element is for buying in the carboxylated long-chain biological element of Thermo Fisher Scientific company of the U.S. (EZ-Link Sulfo-NHS-LC-Biotin, molecular weight 556.59).
The nanometer magnetic bead (30 nm) being modified with Streptavidin is bought in Ocean NanoTech company of the U.S..
Amidized broom molecule is amidized linearizing polyamide-amide, and its molecular weight is 13000 Da, purchased from Weihai Chen Yuan new chemical materials company limited.
Conventional magnetic frame is separated magneticstrength and is less than 30T/m.
N-hydroxysuccinimide 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 800 mL distilled water, adjust pH to 7.4 with 5 M NaOH, then be settled to 1000 mL and namely obtain 0.01 M PBS.Add Tween 20 with the volume ratio of 1/1000 (V/V) again, namely obtain 0.1%PBST.
Embodiment 1
1. broom molecule-antibody complex, prepare in accordance with the following steps:
(1) take the amidized broom molecule of 1.0 mg, be suspended in 4 mL phosphoric acid buffers (PBS, 0.01 mol/L, pH 8.0), the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, makes the final concentration of glutaraldehyde be 3%.Room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min;
(2) methicillin-resistant staphylococcus aureus is dripped to above-mentioned solution
mRSAspecific antibody 1mL, makes its final concentration reach about 3 mg/mL.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, dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize that will obtain.
2. long-chain biological element-broom molecule-antibody complex is prepared in accordance with the following steps:
(1) often get 15 mg long-chain biological elements, be suspended in 4 mL phosphoric acid buffers (PBS, 0.01 mol/L, pH 8.0), the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, makes the final concentration of glutaraldehyde be 3%.Room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min;
(2) 0.53 mg broom molecule-antibody complex is joined in 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, dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize that will obtain.
3. enrichment is caught: get testing sample solution 1 mL, adds 0.1 mg long-chain biological element-broom molecule-antibody complex, is placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm formed long-chain biological element-broom molecule-antibody-
mRSAantigenic compound; Add the nanometer magnetic bead that 0.1 mg is modified with Streptavidin, be placed on blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again, centrifuge tube is inserted conventional magnetic frame and be separated 3 min;
4. after deionized water cleans gently, with PBS damping fluid mix resuspended namely obtain mixture nanometer magnetic bead-Streptavidin-vitamin H-broom molecule-antibody of being enriched with methicillin-resistant staphylococcus aureus-
mRSAantigen.
Embodiment 2 concentration effect is tested
(1) getting 1 mL concentration is 10
4cfu/mL's
mRSAin 1.5 mL sterile centrifugation tube, centrifugal 5 min of 12000 rpm, abandon supernatant, resuspended by the aseptic PBS solution of equal-volume.
(2) enrichment is caught: arrange respectively technical solution of the present invention group (
mRSAantibody and the plain co-modified broom group of molecules of long-chain biological),
mRSAspecific antibody modify nanometer magnetic bead group,
mRSAthe micron magnetic bead group enrichment object bacterium that specific antibody is modified.
(3) after Magneto separate, supernatant liquor is poured in sterile centrifugation tube, and separate and caught
mRSAimmunomagnetic beads then with PBST cleaning twice, to mix, and with the resuspended immunomagnetic beads mixture of the aseptic PBS solution of 1 mL.
(4) capture rate calculates: after the object bacterium re-suspension liquid of each group of enrichment is carried out gradient dilution, count each gradient with flat board, and by the capture rate of capture rate formulae discovery object bacteria, each experiment in triplicate.The calculation formula of each group of capture rate is as follows: (total number of bacterial colony of being adsorbed by enrichment/all total plate count) × 100%.
The scheme that object bacterium is caught in described each group of enrichment is as follows:
A. technical solution of the present invention group (
mRSAantibody and the co-modified broom group of molecules of long-chain biological element) enrichment catches object bacterium scheme as embodiment 1, specific as follows:
By 0.1 mg
mRSAantibody and the co-modified broom molecule of vitamin H and vitamin H-broom molecule-antibody complex join containing in object bacteria centrifuge tube, are placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm
.then add the nanometer magnetic bead that 0.1 mg is modified with Streptavidin, be placed on blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again.Finally, centrifuge tube is inserted conventional magnetic frame and be separated 3 min.
B.
mRSAit is specific as follows that object bacterium scheme is caught in the nanometer magnetic bead group enrichment that specific antibody is modified:
0.1 mg is prepared
mRSAthe nanometer magnetic bead that specific antibody is modified joins containing in object bacteria centrifuge tube, is placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm.Finally, centrifuge tube is inserted conventional magnetic frame and be separated 3 min.
Described
mRSAthe nanometer magnetic bead preparation that specific antibody is modified: (1) is got 10 mg nanometer magnetic beads (30 nm do not have coupling Streptavidin) and used dehydrated alcohol successively, 1 M NaOH, 1 M HCl respectively washs once, PBS(0.02 M, pH 4.0) wash three times, aseptic PBS is resuspended.Add NHSS 0.4 mg, EDC 0.35 mg, be placed on blending instrument and keep magnetic bead to suspend, 37 DEG C of activation 2 h.(2) magnetic frame reclaim magnetic bead, PBS(0.02 M, pH 4.0) washing three times after, magnetic bead is resuspended in aseptic PBS, adds 80 μ g by every mg magnetic bead
mRSAspecific antibody, is placed in 37 DEG C of coupling 2 h on blending instrument.(3) add thanomin room temperature and close 2 h.Magnet stand reclaims magnetic bead, and PBS washs three times, and 10 ml PBS(are containing 0.05% NaN
3, 0.5% BSA, pH 7.4) and resuspended immunomagnetic beads for subsequent use in 4 DEG C of Refrigerator stores.
C.
mRSAit is specific as follows that object bacterium scheme is caught in the micron magnetic bead group enrichment that specific antibody is modified:
0.1 mg is prepared
mRSAthe micron magnetic bead that specific antibody is modified joins containing in object bacteria centrifuge tube, is placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm.Finally, centrifuge tube is inserted conventional magnetic frame and be separated 3 min.
Described
mRSAthe micron magnetic bead preparation that specific antibody is modified: (1) is got 10 mg micron magnetic beads (1150 nm do not have coupling Streptavidin) and used dehydrated alcohol successively, 1 M NaOH, 1 M HCl respectively washs once, PBS(0.02 M, pH 4.0) wash three times, aseptic PBS is resuspended.Add NHSS 0.4 mg, EDC 0.35 mg, be placed on blending instrument and keep magnetic bead to suspend, 37 DEG C of activation 2 h.(2) magnetic frame reclaim magnetic bead, PBS(0.02 M, pH 4.0) washing three times after, magnetic bead is resuspended in aseptic PBS, adds 80 μ g by every mg magnetic bead
mRSAspecific antibody, is placed in 37 DEG C of coupling 2 h on blending instrument.(3) add thanomin room temperature and close 2 h.Magnet stand reclaims magnetic bead, and PBS washs three times, and 10 ml PBS(are containing 0.05% NaN
3, 0.5% BSA, pH 7.4) and resuspended immunomagnetic beads for subsequent use in 4 DEG C of Refrigerator stores.
Each group of capture rate is as follows:
| MRSAThe micron magnetic bead group capture rate that specific antibody is modified | MRSAThe nanometer magnetic bead group capture rate that specific antibody is modified | MRSAAntibody and the plain co-modified broom group of molecules capture rate of long-chain biological |
| 55.9% | 19.6% | 90.3% |
Experimental result shows,
mRSAthe capture rate of the micron magnetic bead group that specific antibody is modified is apparently higher than the capture rate of nanometer magnetic bead group, and this illustrates contrast nanometer magnetic bead group, because micron magnetic bead volume is large, magnetic strong, and 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 again
mRSAthe micron magnetic bead group that specific antibody is modified, this shows that technical solution of the present invention can increase object bacteria nano surface magnetic bead fraction of coverage by broom molecule, thus magnetic is improved greatly, and then achieve (3min) high efficiency separation enrichment methicillin-resistant staphylococcus aureus at short notice.
Experiment is caught in embodiment 3 enrichment
Conventional magnetic frame disengaging time is 30min, and all the other are with embodiment 2.
Each group of capture rate is as follows:
| MRSAThe micron magnetic bead group capture rate that specific antibody is modified | MRSAThe nanometer magnetic bead group capture rate that specific antibody is modified | MRSAAntibody and the plain co-modified broom group of molecules capture rate of long-chain biological |
| 56.7% | 38.9% | 91.2% |
Experimental result shows, is separated 3min in comparative example 2, and when reaching 30min when disengaged, the capture rate of three groups is obtained for raising, particularly
mRSAthe capture rate of the nanometer magnetic bead group that specific antibody is modified improves the most obvious, and this shows to improve the capture rate of nanometer magnetic bead group widely by time expand, but its still lower than short period of time separation (3min) time
mRSAthe capture rate of antibody and the plain co-modified broom group of molecules of long-chain biological.This shows that technical solution of the present invention can (3min) high efficiency separation enrichment methicillin-resistant staphylococcus aureus at short notice.
Embodiment 4
Aseptic meat is pulverized, makes testing sample solution in the usual way, add
mRSAregulate bacterium colony concentration to 10
4cfu/mL is for subsequent use.
By what prepare
mRSAantibody and the co-modified dendrimer (0.1 mg) of long-chain biological element join in sample solution respectively, are placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm
.then add the nanometer magnetic bead (0.1 mg) being modified with Streptavidin, be placed on blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again.Finally, conventional magnetic frame is separated 3 min.After Magneto separate, supernatant liquor is poured in sterile centrifugation tube, and separate and caught
mRSAimmunomagnetic beads then with PBST cleaning twice, to mix, and with the resuspended immunomagnetic beads of the aseptic PBS solution of 1 mL.Capture rate such as embodiment 2 method obtains, and all the other are with embodiment 2.The results are shown in Table 1, show in this programme energy efficiently concentrating sample separation
mRSA.
Embodiment 5
Germ-free milk is sample testing sample solution, adds
mRSAregulate bacterium colony concentration to 10
4cfu/mL.All the other are with embodiment 4
Embodiment 6
Aseptic grates vegetables, makes testing sample solution in the usual way, adds
mRSAregulate bacterium colony concentration to 10
4cfu/mL.All the other are with embodiment 4.
Embodiment 7
Testing sample is aseptic whole blood, adds
mRSAregulate bacterium colony concentration to 10
4cfu/mL.All the other are with embodiment 4.
In the different actual sample of table 2
mRSAthe comparison of separating effect
| Actual sample | MRSAAntibody and the plain co-modified broom group of molecules capture rate of long-chain biological |
| Embodiment 4 meat | 83.2% |
| Embodiment 5 milk | 84.1% |
| Embodiment 6 vegetables | 84.7% |
| Embodiment 7 whole blood | 80.2% |
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (3)
1. methicillin-resistant staphylococcus aureus fast separating process, it is characterized in that comprising the following steps: (1) takes 1.0 mg broom molecules, be suspended in 4 mL 0.01 mol/L pH 8.0 PBS phosphoric acid buffers, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, the final concentration of glutaraldehyde is made to be 3%, room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; Drip 1mL methicillin-resistant staphylococcus aureus
mRSAspecific antibody, makes its final concentration reach about 3 mg/mL, room temperature reaction 24 h under the rotating speed of shaking table 150 r/min; Decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize obtained to obtain broom molecule-antibody complex; (2) 15 mg long-chain biological elements are got, be suspended in 4 mL 0.01mol/L pH 8.0 PBS phosphoric acid buffers, the glutaraldehyde water solution 545 μ L of agitation and dropping 25%, makes the final concentration of glutaraldehyde be 3%, room temperature reaction 3.5 h under the rotating speed of shaking table 150 r/min; Add 0.53 mg broom molecule-antibody complex, room temperature reaction 24 h under the rotating speed of shaking table 150 r/min; Decompression is spin-dried for solvent, deionized water dissolving, and dialyse 1 d in PBS and deionized water; Dialysis terminates the solution lyophilize obtained to obtain long-chain biological element-broom molecule-antibody complex; (3) get 1 mL testing sample solution, add 0.15 mg
mRSAantibody and long-chain biological element co-modified broom molecule and step (2) long-chain biological element-broom molecule-antibody complex, be placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm; Add the nanometer magnetic bead that 0.15 mg is modified with Streptavidin, be placed on blending instrument, with rotating speed incubated at room 15 min of 30 rpm, conventional magnetic frame is separated 3 min; Described nanometer magnetic bead particle diameter is 20-50 nm; (4) after magneticseparation, after deionized water cleans gently, with PBS damping fluid mix resuspended namely obtain nanometer magnetic bead-Streptavidin-long-chain biological element-broom molecule-antibody of being enriched with methicillin-resistant staphylococcus aureus-
mRSAantigenic compound.
2. method according to claim 1, it is characterized in that described broom molecule is amidized linearizing polyamide-amide, its molecular weight is 13000 Da.
3. method according to claim 1, is characterized in that described nanometer magnetic bead particle diameter is 30 nm.
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Granted publication date: 20150415 |