CN106967709A - The method that the magnetic nano-particle fast enriching of antibiotics modification separates Listeria monocytogenes - Google Patents
The method that the magnetic nano-particle fast enriching of antibiotics modification separates Listeria monocytogenes Download PDFInfo
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Abstract
The method that the magnetic nano-particle fast enriching of antibiotics modification separates Listeria monocytogenes.The invention discloses a kind of separation method of pathogenic bacteria, Listeria monocytogenes (Listeria monocytogenes) separation method is related generally to, this method preferably carries out follow-up study for purpose bacterium and provides basis, is related to biological technical field.Method includes the purpose bacterium that magnetic bead is coupled with bovine serum albumin(BSA) (BSA), modified again with vancomycin in the coated bead complexes of bovine serum albumin(BSA), bovine serum albumin(BSA) and the co-modified bead complexes capture sample liquid of vancomycin, by the effect of externally-applied magnetic field, captured object bacteria such as is separated and is resuspended with sample liquid at the step.The purpose bacterium captured by Magneto separate can directly carry out subsequent analysis, compared with traditional bacterium magnetism separate method, this method can carry out Magneto separate to the gram-positive bacteria in food substrate and blood, purpose bacterium separative efficiency in sample be improved, while reducing cost.
Description
Technical field
The present invention relates to biological technical field, the Listeria monocytogenes separation method based on magnetic bead is specifically related to.
Background technology
Listeria monocytogenes are a kind of pathogenic bacteria of generally existing in nature, the food-borne as caused by Listeria monocytogenes
Disease mainly has enterogastritis, central nervous system infection, miscarriage etc..The food of common Listeria monocytogenes pollution has raw meat system
Product, vegetables, milk, tomato etc..The recall rate of Listeria monocytogenes is up in Canada, 100 hamburger retail samples
52%.Because Listeria monocytogenes can still grow at 4 DEG C of refrigerator temperature, potentially hazardous property is set further to increase, serious prestige
Coerce food security.Therefore, set up a kind of efficient, new method of quick detection Listeria monocytogenes and seem particularly urgent.In view of needing
Set up a kind of efficient, quick detection method, the isolation technics of the magnetic nano-particle based on functionalization is monitored in pathogenic bacteria
In developed rapidly.
The content of the invention
For the defect of prior art, it is an object of the invention to provide a kind of capture rate is high, easy, disengaging time is short,
The method of quick from complex matrices under low gradient magnetic, special separation purpose bacterium Listeria monocytogenes.
The fast separating process of Listeria monocytogenes, comprises the following steps：(1) 2mL magnetic bead is drawn, 8mL pH are added to
In the PBS solution of=7.4 sterilizing, then in the presence of externally-applied magnetic field, magnetic bead will be washed, be repeated 3 times, will washed
Good magnetic bead is resuspended in the PBS solution of 10mL sterilizings；(2) in 5.8mg EDC, the PBS for being dissolved in 290 μ L sterilizings, 6.5mg
In NHSS, the PBS for being dissolved in 325 μ L sterilizings, then the EDC and NHSS after dissolving are added in washed magnetic bead, 1h is activated；
(3) magnetic bead after activation is washed 3 times with the PBS of sterilizing, in the PBS solution for being resuspended in 8mL sterilizings；(4) bovine serum albumin is weighed
In white 24mg, the PBS solution for being dissolved into 3mL sterilizings, it is then added in the magnetic bead solution after activation, reacts 3h, then with goes out
The PBS of bacterium is washed 3 times, in the PBS solution for being resuspended in 8mL sterilizings, obtains the magnetic bead of Bovine Serum Albumin Modified；(5) weigh
200mg vancomycins, 169.31mg EDC and 47.98mg NHSS, are dissolved in the PBS solution of sterilizing respectively, and then mixing passes through
Carbodiimide method, activates vancomycin surface carboxyl groups, activates 10min；(6) vancomycin after activation is added to cow's serum
In the magnetic bead of albumin modification, 6h is incubated；(7) after completion of the reaction, washed 3 times with the PBS of sterilizing, be resuspended in 10mL sterilizings
In PBS solution, vancomycin-bovine serum albumin(BSA)-bead complexes that concentration is 2mg/mL are obtained, for being enriched with single increasing Li Si
Special bacterium.(8) 10mL testing sample solutions are taken, 0.7mg vancomycins-bovine serum albumin(BSA)-bead complexes is added, is placed in culture
On case, with 200rpm 37 DEG C of incubation 45min of rotating speed；Insert magnetic frame separation 3min；(9) after Magneto separate, sterile PBS solution is clear
After washing, the Listeria monocytogenes-vancomycin-ox for being enriched with Listeria monocytogenes is produced with mixed be resuspended of sterile PBS
Seralbumin-bead complexes.
The bovine serum albumin(BSA), its molecular weight is 66000Da.
The magnetic bead particle diameter is 180nm, surface carboxyl groups.
Because there are multiple amino on bovine serum albumin(BSA) surface, therefore vancomycin passes through carboxyl and the amino of bovine serum albumin(BSA)
Be connected, can also the amino of bovine serum albumin(BSA) be connected with the carboxyl of magnetic bead surfaces, then vancomycin by five hydrogen bonds and
The D- alanyls on Listeria monocytogenes surface-D-alanine (D-Ala-D-Ala) molecule is connected.
Concrete principle is shown in Fig. 1.
This method is particularly suitable that purpose bacterium is separated from complex matrices suitable for from the separation purpose bacterium in sample substrate.
Food samples include the food material after all kinds of fresh or freeze cutting, such as fresh vegetables, meat, seafood and milk production
Product.According to conventional treatment method such as solution to be measured will be made in sample treatment after sample comminution.
Had the advantages that using technical solution of the present invention：
1st, the present invention uses 180nm magnetic nano-particle, is mainly used in the fast enriching of object bacteria in matrix, and
Prior art is using the amplification that the enrichment method of 30nm or 50nm nanometer magnetic bead combination dendrimers is for magnetic signal.
2nd, the vancomycin that the present invention is used is traditional commercial pharmaceutical, and compared to antibody, with stablizing, cost is low,
Quality controllable the advantages of.Based on above-mentioned advantage, in single separation process, separation vancomycin-cow's serum that the present invention is built
Albumin-magnetic nano particle compound reduces 195 times for older than immune Magneto separate in cost；Compare in terms of material preservation
The immunomagnetic beads shelf-life is also longer.
3rd, it is the environment that various pathogens coexist in food substrate or blood.The vancomycin that the present invention is used is
The molecular recognition agent of the identification gram-positive bacteria of wide spectrum, is a kind of general point for the gram-positive bacteria in isolation medium
From strategy, most of gram-positive bacterias in matrix can all be separated, then pass through PCR (PCR)
Or selectivity culture is differentiated.By contrast, antibody because its can only be single-minded identification, so as to isolate corresponding target
Bacterium, other pathogenic bacteria being present in mechanism can not differentiate.
4th, vancomycin-bovine serum albumin(BSA)-magnetic nano particle compound that the present invention is synthesized is first that ox blood is pure
Then vancomycin modification is arrived bovine serum albumin(BSA)-magnetic nano-particle by albumen coupling again to the surface of magnetic nano-particle
Surface, after being first coupled compared to vancomycin with bovine serum albumin(BSA), then with combining poly-D-lysine (PLL) modified magnetic nanometer
The method of particle, vancomycin-bovine serum albumin(BSA)-magnetic nano particle compound of the invention not only can be up to more preferable point
From efficiency, and in materials synthesis, time-consuming shorter time and cost are lower.
5th, bovine serum albumin(BSA) molecule is first coupled at magnetic nano-particle surface by the present invention, then again by vancomycin point
Son is coupled on the coated magnetic nano-particle of bovine serum albumin(BSA), it is to avoid in conventional method by vancomycin molecule coupling labeled in
Vancomycin molecule is directly directly connected on magnetic nano-particle surface by magnetic bead surfaces, causes vancomycin or mould through the ages
Plain molecular activity reduction and the big shortcoming of steric hindrance.Bovine serum albumin(BSA) has considerable flexibility as molecular arm simultaneously,
Vancomycin molecule is conducive to be connected with D- alanyls-D-alanine (D-Ala-D-Ala) molecule on Listeria monocytogenes surface,
Improve capture rate., should compared to directly coupling or by method of the amino in vancomycin molecule as reactive group
Method separation Listeria monocytogenes ability is stronger, is particularly suitable for use in the separation of complex sample Listeria monocytogenes, such as food samples,
Whole blood sample etc..
6th, it is general to use hydrophobic adsorbent or chemical coupling mode be active during magnetic nano-particle coupling vancomycin
Vancomycin be connected in magnetic nano-particle surface.Vancomycin is too near with magnetic nano-particle surface distance, magnetic Nano
The hydrophobic or strong hydrophilicity group of particle nature and its remained on surface easily causes vancomycin space conformation to change, and leads
Vancomycin bioactivity is caused to decline.But this experimental program introduces bovine serum albumin(BSA) molecule in coupling process, it has
Certain space length, so that vancomycin molecule is away from magnetic nano-particle and magnetic nano-particle surface, it is to avoid magnetic
The influence of property nano-particle nature and surface to vancomycin molecule.Meanwhile, bovine serum albumin(BSA) is introduced without influence
The space conformation of vancomycin molecule, so as to serve the effect of protection vancomycin molecular biological activity.
7th, the present invention uses bovine serum albumin(BSA) molecule, improves stability of the magnetic nano-particle in solution, it is to avoid occur
Precipitation, increases the water solubility and biocompatibility of compound.
Brief description of the drawings
The operational flowchart of magnetic separation technique involved in the present invention Fig. 1；
Embodiment
In order that the present invention is more clearly understood, with reference to embodiments, the present invention will be described in further detail.Should
Work as understanding, specific embodiment described herein only to explain the present invention, is not intended to limit the present invention.
Magnetic bead (180nm) is bought in Shanghai Ao Run Co., Ltds.
Bovine serum albumin(BSA) 66000Da, purchased from Weihai Chen Yuan new chemical materialses Co., Ltd.
Vancomycin molecule is purchased from Shanghai Aladdin Co., Ltd.
N- hydroxysuccinimide NHSS, ethyl 3- (3- dimethylaminos) carbodiimide hydrochloride EDC etc. is conventional examination
Agent, is repeated no more.
0.01M PBS compound methods：8.0g NaCl、0.2g KCl、0.24g KH2PO4、1.44g Na2HPO4It is dissolved in
In 800mL distilled water, pH is adjusted to 7.4 with 5M NaOH, then be settled to 1000mL and produce 0.01M PBS.
Embodiment 1
1. vancomycin-bovine serum albumin(BSA)-bead complexes, is prepared in accordance with the following steps:
(1) 2mg magnetic beads are taken, are suspended in 8mL phosphate buffers (PBS, 0.01mol/L, pH 7.4), are washed 3 times, plus
In 5.8mg EDC, the PBS for being dissolved in 290 μ L sterilizings, 6.5mg NHSS are dissolved in the PBS that 325 μ L sterilize, then by after dissolving
EDC and NHSS are added in washed magnetic bead, activate 1h；；
(2) bovine serum albumin(BSA) 24mg is weighed, in the PBS solution for being dissolved into 3mL sterilizings, the magnetic after activation is then added to
In pearl solution, 2h is reacted, is then washed 3 times with the PBS of sterilizing, in the PBS solution for being resuspended in 8mL sterilizings, ox blood is obtained pure
Protein modified magnetic bead；
(3) 200mg vancomycins are weighed, 169.31mg EDC and 47.98mg NHSS are dissolved in the PBS solution of sterilizing respectively
In, then mixing activates vancomycin surface carboxyl groups by carbodiimide method, activates 10min；By the vancomycin after activation
It is added in the magnetic bead of Bovine Serum Albumin Modified, is incubated 6h；
(4) after completion of the reaction, washed 3 times with the PBS of sterilizing, in the PBS solution for being resuspended in 10mL sterilizings, obtaining concentration is
2mg/mL vancomycin-bovine serum albumin(BSA)-bead complexes, for being enriched with Listeria monocytogenes.
2. enrichment capture：Testing sample solution 10mL is taken, 0.7mg vancomycins-bovine serum albumin(BSA)-magnetic bead is added and is combined
Thing, is placed on blending instrument, pure with 200rpm rotating speed incubation at room temperature 45min formation Listeria monocytogenes-vancomycin-ox bloods
Albumen-bead complexes compound；By centrifuge tube insertion conventional magnetic frame separation 3min；
3. after deionized water is gently cleaned, single increasing Lee for being enriched with Listeria monocytogenes is produced with mixed be resuspended of PBS
This special bacterium-vancomycin-bovine serum albumin(BSA)-bead complexes.
The concentration effect of embodiment 2 is tested
(1) it is 10 to take 1mL concentration5CFU/mL Listeria monocytogenes bacterium in 1.5mL sterile centrifugation tubes, 12000rpm from
Heart 5min, abandons supernatant, is resuspended with isometric sterile PBS solution.
(2) enrichment capture：Technical solution of the present invention group is set respectively：Bovine serum albumin(BSA)-magnetic bead group, vancomycin-magnetic
Pearl, vancomycin-bovine serum albumin(BSA)-magnetic bead group enrichment purpose bacterium.
(3) after Magneto separate, supernatant is poured into sterile centrifugation tube, and separating capture has the ten thousand of Listeria monocytogenes
Ancient mycin-bovine serum albumin(BSA)-magnetic bead then with PBS twice, be well mixed, and single increasing Lee is resuspended with the sterile PBS solutions of 1mL
This special bacterium-vancomycin-bovine serum albumin(BSA)-magnetic bead.
(4) capture rate is calculated：The purpose bacterium re-suspension liquid that each group is enriched with is carried out after gradient dilution, with flat board to each gradient
Count, the capture rate of object bacteria is calculated by capture rate formula, test every time in triplicate.The calculating of each group capture rate
Formula is as follows：[by the total plate count of enrichment absorption/(and total plate count in supernatant+total plate count adsorbed by enrichment)] ×
100%.
The scheme of each group enrichment capture purpose bacterium is as follows：
A. technical solution of the present invention group (vancomycin-bovine serum albumin(BSA)-magnetic bead group) enrichment captures purpose bacterium scheme such as
Embodiment 1, it is specific as follows：
0.7mg vancomycins-bovine serum albumin(BSA)-bead complexes are added in centrifuge tube containing object bacteria, are placed in mixed
On even instrument, 45min is incubated at room temperature with 200rpm rotating speed.By centrifuge tube insertion conventional magnetic frame separation 3min.
B. vancomycin-magnetic bead group enrichment capture purpose bacterium scheme is specific as follows：
0.7mg vancomycin-the magnetic beads prepared are added in centrifuge tube containing object bacteria, are placed on blending instrument, with
200rpm rotating speed incubation at room temperature 45min.Finally, centrifuge tube is inserted into conventional magnetic frame and separates 3min.
It is prepared by the magnetic bead of the vancomycin modification：(1) 2mL magnetic bead is taken, the sterile of 8mL pH=7.4 is added to
In PBS solution, then in the presence of externally-applied magnetic field, magnetic bead will be washed, be repeated 3 times, the magnetic bead washed will be resuspended
In the sterile PBS solutions of 10mL；(2) 5.8mg EDC, are dissolved in the sterile PBS of 290 μ L, 6.5mg NHSS, be dissolved in 325 μ L without
In the PBS of bacterium, then the EDC and NHSS after dissolving are added in washed magnetic bead, 1h is activated；(3) magnetic bead after activating
Washed 3 times, be resuspended in the sterile PBS solutions of 8mL with sterile PBS；(4) 200mg vancomycins are weighed and are dissolved in sterile PBS
In solution, the magnetic bead then activated is incubated 2h；(7) after completion of the reaction, washed 3 times with sterile PBS, be resuspended in 10mL sterile
PBS solution in, obtain vancomycin-bovine serum albumin(BSA)-bead complexes that concentration is 2mg/mL, single Lee increased for being enriched with
This special bacterium.
C. bovine serum albumin(BSA)-magnetic bead group
(1) 2mL magnetic beads are taken, are suspended in 8mL phosphate buffers (PBS, 0.01mol/L, pH 7.4), are washed 3 times, plus
In 5.8mg EDC, the PBS for being dissolved in 290 μ L sterilizings, 6.5mg NHSS are dissolved in the PBS that 325 μ L sterilize, then by after dissolving
EDC and NHSS are added in washed magnetic bead, activate 1h；
(2) bovine serum albumin(BSA) 24mg is weighed, in the PBS solution for being dissolved into 3mL sterilizings, the magnetic after activation is then added to
In pearl solution, 2h is reacted, is then washed 3 times with the PBS of sterilizing, in the PBS solution for being resuspended in 8mL sterilizings, ox blood is obtained pure
Protein modified magnetic bead；
(3) magnet stand reclaims bovine serum albumin(BSA)-magnetic bead, and PBS is washed three times, 10mL PBS (pH=7.4), and obtaining concentration is
2mg/mL vancomycin-bovine serum albumin(BSA)-bead complexes, for being enriched with Listeria monocytogenes.
Each group capture rate is as follows：
Test result indicates that, the capture rate of vancomycin-bovine serum albumin(BSA)-magnetic bead group is repaiied apparently higher than vancomycin
The magnetic bead group capture rate of decorations, this explanation bovine serum albumin(BSA) adds the combination of vancomycin and object bacteria as carrier molecule
Chance so that the surface of object bacteria combines more nano-particles, therefore, in a short time just can the more mesh of separation and concentration
Mark bacterium.The capture rate of bovine serum albumin(BSA)-magnetic bead group is very low, illustrates bovine serum albumin(BSA) while can also reduce non-spy well
Opposite sex absorption.Three groups of experimental datas more than, illustrate using bovine serum albumin(BSA) as molecule, can not only improve separation effect
Rate, while can also reduce non-specific adsorption well.Explanation, which captures object bacteria with vancomycin molecule, has good effect
Really.
Embodiment 3
Aseptic juice is sample testing sample solution, adds Listeria monocytogenes and adjusts bacterium colony concentration to 105CFU/mL is standby
With.
Vancomycin-bovine serum albumin(BSA)-magnetic bead (0.7mg) will be prepared to be added separately in sample solution, be placed in mixed
On even instrument, 45min is incubated at room temperature with 200rpm rotating speed.Then conventional magnetic frame separates 3min.After Magneto separate, supernatant is fallen
Enter in sterile centrifugation tube, and separating capture has Listeria monocytogenes-Gu mycin-bovine serum albumin(BSA)-bead complexes then
With sterile PBS twice, it is well mixed, and Listeria monocytogenes-vancomycin-cow's serum is resuspended with the sterile PBS solutions of 1mL
Albumin-bead complexes.The capture rate such as method of embodiment 2 is obtained, remaining be the same as Example 2.1 is the results are shown in Table, shows this programme
Listeria monocytogenes in energy efficiently concentrating separation sample.
Embodiment 4
Germ-free milk is sample testing sample solution, adds Listeria monocytogenes and adjusts bacterium colony concentration to 105CFU/mL.Its
Remaining be the same as Example 3.
Embodiment 5
Sterile drinking water is sample testing sample solution, adds Listeria monocytogenes and adjusts bacterium colony concentration to 105CFU/mL。
Embodiment 6
Testing sample is sterile whole blood, adds Listeria monocytogenes and adjusts bacterium colony concentration to 105CFU/mL.Remaining is with implementation
Example 3.
The comparison of Listeria monocytogenes separating effect in the different actual samples of table 1
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Any modifications, equivalent substitutions and improvements made within principle etc., should be included in the scope of the protection.
Claims (3)
1. the fast separating process of Listeria monocytogenes, it is characterised in that comprise the following steps：(1) 2mL magnetic bead is drawn, is added
Into the PBS solution of 8mL pH=7.4 sterilizing, then in the presence of externally-applied magnetic field, magnetic bead is washed, is repeated 3 times,
The magnetic bead washed is resuspended in the PBS solution of 10mL sterilizings；(2) in 5.8mg EDC, the PBS for being dissolved in 290 μ L sterilizings, 6.5mg
In NHSS, the PBS for being dissolved in 325 μ L sterilizings, then the EDC and NHSS after dissolving are added in washed magnetic bead, 1h is activated；
(3) magnetic bead after activation is washed 3 times with the PBS of sterilizing, in the PBS solution for being resuspended in 8mL sterilizings；(4) bovine serum albumin is weighed
In white 24mg, the PBS solution for being dissolved into 3mL sterilizings, it is then added in the magnetic bead solution after activation, reacts 2h, then with goes out
The PBS of bacterium is washed 3 times, in the PBS solution for being resuspended in 8mL sterilizings, obtains the magnetic bead of Bovine Serum Albumin Modified；(5) weigh
200mg vancomycins, 169.31mg EDC and 47.98mg NHSS, are dissolved in the PBS solution of sterilizing respectively, and then mixing passes through
Carbodiimide method, activates vancomycin surface carboxyl groups, activates 10min；(6) vancomycin after activation is added to cow's serum
In the magnetic bead of albumin modification, 6h is incubated；(7) after completion of the reaction, washed 3 times with the PBS of sterilizing, be resuspended in 10mL sterilizings
In PBS solution, vancomycin-bovine serum albumin(BSA)-bead complexes that concentration is 2mg/mL are obtained, for being enriched with single increasing Li Si
Special bacterium；(8) 10mL testing sample solutions are taken, 0.7mg vancomycins-bovine serum albumin(BSA)-bead complexes is added, is placed in culture
On case, with 200rpm 37 DEG C of incubation 45min of rotating speed；Insert magnetic frame separation 3min；(9) after Magneto separate, sterile PBS solution is clear
After washing, the Listeria monocytogenes-vancomycin-ox for being enriched with Listeria monocytogenes is produced with mixed be resuspended of sterile PBS
Seralbumin-bead complexes.
2. according to the method described in claim 1, it is characterised in that described its molecular weight of bovine serum albumin(BSA) molecule is
66000Da。
3. according to the method described in claim 1, it is characterised in that the magnetic bead particle diameter is 180nm, the magnetic of surface modification carboxyl
Pearl.
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| CN110527640A (en) * | 2018-05-25 | 2019-12-03 | 中国农业科学院北京畜牧兽医研究所 | A method of based on cellulose magnetic nanoparticle enrichment and separation anaerobism Fibrolytic bacteria |
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| CN109470853A (en) * | 2017-09-08 | 2019-03-15 | 广州市丹蓝生物科技有限公司 | Diagnosing autoimmune diseases liquid phase protein chip, kit and production method |
| CN110527640A (en) * | 2018-05-25 | 2019-12-03 | 中国农业科学院北京畜牧兽医研究所 | A method of based on cellulose magnetic nanoparticle enrichment and separation anaerobism Fibrolytic bacteria |
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Application publication date: 20170721 |