CN103305441A - High-efficiency rapid vibrio parahemolyticus enrichment and separation method - Google Patents
High-efficiency rapid vibrio parahemolyticus enrichment and separation method Download PDFInfo
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Abstract
The invention discloses a high-efficiency rapid enrichment and separation method, mainly relates to a method for enriching and separating vibrio parahemolyticus from a matrix, aims at providing basis for follow-up study on target bacteria, and relates to the field of biotechnology. The method comprises the following steps of: carrying out covalent coupling on an arborescence hyper-branched polymer and an antibody, enveloping the antibody-modified arborescence hyper-branched polymer with long-chain biotin molecules, capturing the target bacteria in a sample liquid by using the arborescence hyper-branched polymer which is co-modified by the antibody and the long-chain biotin, recognizing and coupling the long-chain biotin arborescence hyper-branched polymer in the sample liquid by using streptavidin-modified nano magnetic beads, separating and heavy-suspending the captured bacteria, and the like. The captured bacteria can be subjected to follow-up analysis directly; and compared with the conventional bacterium separation method, the method disclosed by the invention is more applicable to magnetic separation of bacteria in a complex matrix, and the target bacterium separation efficiency in the sample is improved.
Description
Technical field
The present invention relates to biological technical field, specifically relate to the food-borne pathogens separation method based on nanometer magnetic bead.
Background technology
Vibrio parahemolyticus (
Vibrio parahaemolyticus,VP), Vibrio is the polymorphic bacterium that a kind of Grain-negative is had a liking for salt.Be distributed widely in the sea-foods such as inshore seawater, seabed silt, planktonic organism and fish, shrimp, shellfish.Can cause acute gastroenteritis and primary septicemia, according to Ministry of Health report, the food poisoning that Vibrio parahemolyticus causes in the second quarter in 2008 whole nation food poisoning situation occupies the 2nd of microorganism property food poisoning, is only second to Bacillus cereus.The novel method of setting up a kind of efficient rapid detection Vibrio parahemolyticus seems particularly urgent, in view of needs set up a kind of efficient, detection method fast, immune magnetic separation technique has obtained developing rapidly in Surveillance for foodborne pathogen.
The immunity magnetic separation technique is one of important component part of food-borne pathogens rapid screening technology, but object bacteria in this technology efficient capture, concentrated enrichment liquid, improves pathogenic microbes detect 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 then will be connected with antibody drop in the sample liquid to object bacteria catch, enrichment, magnetic separates (concrete principle is seen Fig. 2 A).Yet, should there be many limitation based on the isolation technique of micron order immunomagnetic beads at present: 1) the specific surface area less of micron magnetic bead, reduced magnetic capture efficient; 2) because the particle properties of micron magnetic bead self, by heterogeneous reaction (multiphase reaction) combination, usually need the longer time go specificity to catch bacterial cell in the food substrate between itself and the bacterial cell; 3) micron magnetic bead monodispersity is relatively poor, and precipitation easily occurs self to assemble or form in food substrate liquid; 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 miscellaneous bacteria concentration wherein non-purpose pathogenic bacterium are large, the micron magnetic bead easily produces 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 that the activated antibody of tool is connected in magnetic bead surfaces.Antibody and magnetic bead surfaces distance are too near, and the hydrophobic or strong hydrophilicity group of magnetic bead nature and remained on surface thereof causes that easily the antibody space conformation changes, and cause the antibody biological activity to descend.
Summary of the invention
For the defective of prior art, the purpose of this invention is to provide that a kind of capture rate is high, easy, disengaging time is short, the method for (less than 30 T/m) quick from the food substrate of complexity, special separation purpose bacterium Vibrio parahemolyticus under the low gradient magnetic.
A kind of efficiently concentration and separation Vibrio parahemolyticus method fast may further comprise the steps:
(1) whenever gets the tree-shaped over-expense polymer dissolution of 1 mg in 2 mL, 0.02 M, pH 6.5 phosphoric acid buffer PBS add 0.6 mg N-maloyl imines NHSS, 0.4 mg ethyl 3-(3-dimethylamino) carbodiimide hydrochloride EDC, room temperature places on the blending instrument and stirs, and activates 15 min; Add 2.68 mg Vibrio parahemolyticus specific antibodies, room temperature places and stirs 30 min on the blending instrument; Decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize that obtains is got tree-shaped over-expense polymkeric substance-antibody complex; (2) getting 15 mg long-chain vitamin Hs, 3.6 mgN-maloyl imines NHSS, 2.4 mg ethyl 3-(3-dimethylaminos) carbodiimide hydrochloride EDC is dissolved in 2 mL, 0.02 M pH, the 6.5 PBS damping fluids; Add the tree-shaped over-expense polymkeric substance-antibody complex of 2.24 mg, room temperature places and stirs 30 min on the blending instrument; Decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize that obtains is got long-chain vitamin H-tree-shaped over-expense polymkeric substance-antibody complex; (3) get 1 mL testing sample solution, adding the co-modified tree-shaped over-expense polymkeric substance of 0.1 mg Vibrio parahemolyticus antibody and long-chain vitamin H is long-chain vitamin H-tree-shaped over-expense polymkeric substance-antibody complex, place on the blending 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 blending instrument, with rotating speed incubated at room 15 min of 30 rpm, conventional magnetic frame separates 3 min; (4) after the magneticseparation, with after the 0.1% PBST washing, with the resuspended nanometer magnetic bead-Streptavidin of the catching Vibrio parahemolyticus-long-chain vitamin H-tree-shaped over-expense polymkeric substance-antibody-Vibrio parahemolyticus antigenic compound that namely gets of PBS damping fluid.
Described tree-shaped over-expense polymkeric substance is for being amidized dendroid polyamide-amide, and its molecular weight is 56000 Da.Structure such as Fig. 1.
Described nanometer magnetic bead particle diameter is 20-50nm, is preferably 30 nm.
Tree-shaped over-expense polymkeric substance is realized the covalent coupling of tree-shaped over-expense polymkeric substance and antibody by the carboxyl of amino and Vibrio parahemolyticus specific antibody.
Tree-shaped over-expense polymkeric substance is realized the covalent coupling of tree-shaped over-expense polymkeric substance and long-chain vitamin H by the carboxyl of amino and long-chain biotin molecule; Add excessive long-chain vitamin H to guarantee exposed amino sites on the tree-shaped over-expense polymkeric substance of sealing.
Concrete principle is seen Fig. 2 B.
Present method is applicable to the separation of purpose bacterium in the sample, particularly complex matrices, such as food samples, whole blood sample etc.Food samples comprises the food material after all kinds of fresh or freezing processing, such as products such as fresh vegetables, meat, seafood and milks.Sample preparation is processed according to national standard method and is got final product, as making solution to be measured behind the sample sterile crushing.
Adopt technical solution of the present invention to have following beneficial effect:
1, the present invention is by the cascade scale effect of tree-shaped over-expense polymkeric substance, magnetic bacterium signal exponentially level is enlarged, under lower magneticstrength, just can realize the separation of magnetic bacterium, and within the identical time, compare than routine immunization magnetic bead separation method, be separated to purpose bacterium ability stronger, be specially adapted to the separation of complex sample, such as food samples, whole blood sample etc., magnetic field demanding defective slow for the purpose bacterium speed in the 20-50 nm immunomagnetic beads separate complex matrix sample behind the simple employing antibody modification, adopt tree-shaped over-expense polymkeric substance to realize the amplification of nanometer magnetic bead magnetic signal, thereby improved purpose bacterium separation efficiency in the complex matrices sample, realized purpose bacterium specificity sharp separation in (less than 30 T/m) are complicated under low gradient magnetic the food substrate.
2, this programme has been avoided in the ordinary method antibody molecule being coupled to magnetic bead surfaces and has been caused antibody activity to reduce and sterically hindered large shortcoming for antibody molecule is coupled on the tree-shaped over-expense polymkeric substance.
3, the present invention adopts tree-shaped over-expense polymkeric substance, can make reaction soln 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 tree-shaped over-expense polymkeric substance, the nanometer magnetic bead that can modify in conjunction with Streptavidin, thus make on the tree-shaped over-expense polymkeric substance 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 large, 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 sepn process, introduced tree-shaped over-expense polymkeric substance, be connected with a large amount of long-chain biotin molecules on the tree-shaped over-expense polymkeric substance, 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 tree-shaped over-expense polymkeric substance in conjunction with a large amount of nanometer magnetic beads, greatly increase the magnetic bead quantity of target bacteria surface bonding, realized the target bacteria that sharp separation is caught under magnetic field.Comparing with traditional bacterial magnetic separation method, is nanometer magnetic bead more stable in matrix because of what add, and the method more is applicable in complex matrices bacterium to be 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 that the activated antibody of tool is connected in magnetic bead surfaces.Antibody and magnetic bead surfaces distance are too near, and the hydrophobic or strong hydrophilicity group of magnetic bead nature and remained on surface thereof causes that easily the antibody space conformation changes, and cause the antibody biological activity to descend.Yet this experimental program is introduced tree-shaped over-expense polymkeric substance in coupling process, it has certain space size, thereby makes antibody molecule away from magnetic bead and magnetic bead surfaces, has avoided the disadvantageous effect of magnetic bead nature and surperficial antagonist molecule.Simultaneously, the tree-shaped over-expense polymkeric substance of introducing but can not affect the antibody space conformation, thereby has played the bioactive effect of protection antibody molecule.
Description of drawings
The structural representation of the tree-shaped over-expense polymkeric substance 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 explain the present invention, is not intended to limit the present invention.
The long-chain vitamin H is for buying in the carboxylated long-chain vitamin H 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..
Amidized tree-shaped over-expense polymkeric substance is amidized dendroid polyamide-amide, and its molecular weight is 56000 Da, available from Weihai Chen Yuan new chemical materials company limited.
Conventional magnetic frame separates magneticstrength 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 again 1000 mL 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. tree-shaped over-expense polymkeric substance-antibody complex, in accordance with the following steps preparation:
(1) whenever get the tree-shaped over-expense polymer dissolution of 1 mg in 2 mL, 0.02 M, pH 6.5 PBS add 0.6 mg NHSS, 0.4 mg EDC, room temperature places on the blending instrument and stirs, and activates 15 min;
(2) get 2.68 mg Vibrio parahemolyticus specific antibodies and add in the above-mentioned reaction solution, room temperature places and stirs 30 min on the blending instrument;
(3) the mentioned solution decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize that obtains.
2. long-chain vitamin H-tree-shaped over-expense polymkeric substance-antibody complex prepares in accordance with the following steps:
(1) whenever get 15 mg long-chain vitamin Hs, 3.6 mg NHSS, 2.4 mg EDC are dissolved in 2 mL, 0.02 M pH, the 6.5 PBS damping fluids;
(2) the tree-shaped over-expense polymkeric substance-antibody complex of 2.24 mg is joined in the mentioned solution, room temperature places and stirs 30 min on the blending instrument;
(3) the mentioned solution decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize that obtains.
3. enrichment is caught: get testing sample solution 1mL, add 0.1 mg long-chain vitamin H-tree-shaped over-expense polymkeric substance-antibody complex, place on the blending instrument, form long-chain vitamin H-tree-shaped over-expense polymkeric substance-antibody-Vibrio parahemolyticus antigenic compound 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 blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again, centrifuge tube is inserted conventional magnetic frame separate 3 min;
4. after deionized water cleans gently, with mixed resuspended mixture nanometer magnetic bead-Streptavidin of the being enriched with Vibrio parahemolyticus-vitamin H-tree-shaped over-expense polymkeric substance-antibody-Vibrio parahemolyticus antigen that namely gets of PBS damping fluid.
The experiment of embodiment 2 concentration effects
(1) getting 1 mL concentration is 10
4The Vibrio parahemolyticus 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 tree-shaped over-expense polymkeric substance group that Vibrio parahemolyticus antibody and long-chain vitamin H are co-modified), the nanometer magnetic bead group that the Vibrio parahemolyticus specific antibody is modified, the micron magnetic bead group enrichment purpose bacterium that the Vibrio parahemolyticus specific antibody is modified are set respectively.
(3) after magnetic separates, supernatant liquor is poured in the aseptic centrifuge tube, separated the immunomagnetic beads of catching Vibrio parahemolyticus and then clean twice with PBST, mix, and with the resuspended immunomagnetic beads mixture 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 with dull and stereotyped by the capture rate formula, test triplicate at every turn.Each calculation formula of organizing capture rate is as follows: (total number of bacterial colony of being adsorbed by enrichment/all total plate count) * 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 tree-shaped over-expense polymkeric substance group that Vibrio parahemolyticus antibody and long-chain vitamin H are co-modified) enrichment, and be specific as follows:
Be that vitamin H-tree-shaped over-expense polymkeric substance-antibody complex joins and contains in the object bacteria centrifuge tube with 0.1 mg Vibrio parahemolyticus antibody and the co-modified tree-shaped over-expense polymkeric substance of vitamin H, place on the blending instrument, with rotating speed incubated at room 15 min of 30 rpm.Then add 0.1 mg and be modified with the nanometer magnetic bead of Streptavidin, place on the blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again.At last, centrifuge tube is inserted conventional magnetic 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 Vibrio parahemolyticus specific antibody modification:
The nanometer magnetic bead that the Vibrio parahemolyticus specific antibody that 0.1 mg is prepared is modified joins and contains in the object bacteria centrifuge tube, places on the blending instrument, with rotating speed incubated at room 15 min of 30 rpm.At last, centrifuge tube is inserted conventional magnetic frame and separate 3 min.
The nanometer magnetic bead preparation that described Vibrio parahemolyticus specific antibody is modified: (1) is got 10 mg nanometer magnetic beads (30 nm do not have the coupling Streptavidin) and is used successively dehydrated alcohol, 1 M NaOH, each washing of 1 M HCl once, PBS(0.02 M, pH 4.0) wash three times, aseptic PBS is resuspended.Add NHSS 0.4 mg, EDC 0.35 mg places to keep magnetic bead to suspend on the blending instrument 37 ℃ of activation 2 h.(2) magnetic 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 Vibrio parahemolyticus specific antibodies by every mg magnetic bead, places 37 ℃ of coupling 2 h on the blending instrument.(3) add the thanomin 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 for subsequent use in 4 ℃ of Refrigerator stores.
C. it is specific as follows that purpose bacterium scheme is caught in the micron magnetic bead group enrichment of Vibrio parahemolyticus specific antibody modification:
The micron magnetic bead that the Vibrio parahemolyticus specific antibody that 0.1 mg is prepared is modified joins and contains in the object bacteria centrifuge tube, places on the blending instrument, with rotating speed incubated at room 15 min of 30 rpm.At last, centrifuge tube is inserted conventional magnetic frame and separate 3 min.
The micron magnetic bead preparation that described Vibrio parahemolyticus specific antibody is modified: (1) is got 10 mg micron magnetic beads (1150 nm do not have the coupling Streptavidin) and is used successively dehydrated alcohol, 1 M NaOH, each washing of 1 M HCl once, PBS(0.02 M, pH 4.0) wash three times, aseptic PBS is resuspended.Add NHSS 0.4 mg, EDC 0.35 mg places to keep magnetic bead to suspend on the blending instrument 37 ℃ of activation 2 h.(2) magnetic 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 Vibrio parahemolyticus specific antibodies by every mg magnetic bead, places 37 ℃ of coupling 2 h on the blending instrument.(3) add the thanomin 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 for subsequent use in 4 ℃ of Refrigerator stores.
It is as follows that each organizes capture rate:
| The micron magnetic bead group capture rate that the Vibrio parahemolyticus specific antibody is modified | The nanometer magnetic bead group capture rate that the Vibrio parahemolyticus specific antibody is modified | The tree-shaped over-expense polymkeric substance group capture rate that Vibrio parahemolyticus antibody and long-chain vitamin H are co-modified |
| 56.1% | 25.2% | 90.3% |
Experimental result shows, the capture rate of the micron magnetic bead group that the Vibrio parahemolyticus 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 large, 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 again the micron magnetic bead group that the Vibrio parahemolyticus 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 tree-shaped over-expense polymkeric substance, thereby magnetic is improved greatly, and then realized at short notice (3min) high efficiency separation enrichment Vibrio parahemolyticus.
Experiment is caught in embodiment 3 enrichments
Conventional magnetic 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 Vibrio parahemolyticus specific antibody is modified | The nanometer magnetic bead group capture rate that the Vibrio parahemolyticus specific antibody is modified | The tree-shaped over-expense polymkeric substance group capture rate that Vibrio parahemolyticus antibody and long-chain vitamin H are co-modified |
| 58.7% | 39.1% | 92.3% |
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 Vibrio parahemolyticus specific antibody modification improves the most obvious, this shows the capture rate that can improve widely the nanometer magnetic bead group by time expand, but it still is lower than the capture rate of short period of time co-modified tree-shaped over-expense polymkeric substance group of Vibrio parahemolyticus antibody and long-chain vitamin H when separating (3min).This shows at short notice (3min) high efficiency separation enrichment Vibrio parahemolyticus of technical solution of the present invention.
Embodiment 4
Aseptic meat is pulverized, made in the usual way testing sample solution, add Vibrio parahemolyticus and regulate bacterium colony concentration to 10
4Cfu/mL is for subsequent use.
The Vibrio parahemolyticus antibody and the co-modified tree-shaped over-expense polymkeric substance (0.1 mg) of long-chain vitamin H that prepare are joined respectively in the sample solution, place on the blending instrument, with rotating speed incubated at room 15 min of 30 rpm.Then add the nanometer magnetic bead (0.1 mg) be modified with Streptavidin, place on the blending instrument, with the rotating speed of 30 rpm incubated at room 15 min again.At last, conventional magnetic frame separates 3 min.After magnetic separates, supernatant liquor is poured in the aseptic centrifuge tube, separated the immunomagnetic beads of catching Vibrio parahemolyticus 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 Vibrio parahemolyticus in this programme energy efficiently concentrating sample separation.
Embodiment 5
Germ-free milk is the sample testing sample solution, adds Vibrio parahemolyticus 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 Vibrio parahemolyticus and regulates bacterium colony concentration to 10
4Cfu/mL.All the other are with embodiment 4.
Embodiment 7
Testing sample is aseptic whole blood, adds Vibrio parahemolyticus and regulates bacterium colony concentration to 10
4Cfu/mL.All the other are with implementing 4.
The comparison of Vibrio parahemolyticus separating effect in the different actual samples of table 1
| Actual sample | The tree-shaped over-expense polymkeric substance group capture rate that Vibrio parahemolyticus antibody and long-chain vitamin H are co-modified |
| Embodiment 2 meats | 84.4% |
| Embodiment 3 milk | 87.2% |
| Embodiment 4 vegetables | 89.3% |
| Embodiment 5 whole bloods | 83.6% |
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. efficient concentration and separation Vibrio parahemolyticus method fast is characterized in that may further comprise the steps:
(1) whenever gets the tree-shaped over-expense polymer dissolution of 1 mg in 2 mL, 0.02 M, pH 6.5 phosphoric acid buffer PBS add 0.6 mg N-maloyl imines NHSS, 0.4 mg ethyl 3-(3-dimethylamino) carbodiimide hydrochloride EDC, room temperature places on the blending instrument and stirs, and activates 15 min; Add 2.68 mg Vibrio parahemolyticus specific antibodies, room temperature places and stirs 30 min on the blending instrument; Decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize that obtains is got tree-shaped over-expense polymkeric substance-antibody complex; (2) getting 15 mg long-chain vitamin Hs, 3.6 mgN-maloyl imines NHSS, 2.4 mg ethyl 3-(3-dimethylaminos) carbodiimide hydrochloride EDC is dissolved in 2 mL, 0.02 M pH, the 6.5 PBS damping fluids; Add the tree-shaped over-expense polymkeric substance-antibody complex of 2.24 mg, room temperature places and stirs 30 min on the blending instrument; Decompression is spin-dried for solvent, deionized water dissolving, dialysis 1 d in PBS and deionized water; Dialysis finishes the solution lyophilize that obtains is got long-chain vitamin H-tree-shaped over-expense polymkeric substance-antibody complex; (3) get 1 mL testing sample solution, adding the co-modified tree-shaped over-expense polymkeric substance of 0.1 mg Vibrio parahemolyticus antibody and long-chain vitamin H is long-chain vitamin H-tree-shaped over-expense polymkeric substance-antibody complex, place on the blending 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 blending instrument, with rotating speed incubated at room 15 min of 30 rpm, conventional magnetic frame separates 3 min; (4) after the magneticseparation, with after 0.1% PBST (the 0.1% Tween 20) washing, with the resuspended nanometer magnetic bead-Streptavidin of the catching Vibrio parahemolyticus-long-chain vitamin H-tree-shaped over-expense polymkeric substance-antibody-Vibrio parahemolyticus antigenic compound that namely gets of PBS damping fluid.
2. method according to claim 1 is characterized in that described tree-shaped over-expense polymkeric substance for being amidized dendroid polyamide-amide, and its molecular weight is 56000 Da.
3. method according to claim 1 is characterized in that described nanometer magnetic bead particle diameter is 20-50nm, is preferably 30 nm.
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| CN108753769A (en) * | 2018-05-25 | 2018-11-06 | 吉林大学 | The polypeptide and application thereof of vibrio parahemolyticus specific binding |
| CN108753769B (en) * | 2018-05-25 | 2021-08-20 | 吉林大学 | Vibrio parahaemolyticus specific binding polypeptide and application thereof |
| CN112760356A (en) * | 2020-12-23 | 2021-05-07 | 浙江农林大学 | Vibrio parahaemolyticus detection method |
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