CN104313130B - A kind of functionalized magnetic nano particle of efficiently concentrating microorganism and preparation and application - Google Patents
A kind of functionalized magnetic nano particle of efficiently concentrating microorganism and preparation and application Download PDFInfo
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- CN104313130B CN104313130B CN201410490371.2A CN201410490371A CN104313130B CN 104313130 B CN104313130 B CN 104313130B CN 201410490371 A CN201410490371 A CN 201410490371A CN 104313130 B CN104313130 B CN 104313130B
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Abstract
The present invention discloses functionalized magnetic nano particle and the preparation and application of a kind of efficiently concentrating microorganism.The method for being carried out efficiently concentrating and detection of nucleic acids to the microorganism in food based on the functionalized magnetic nano particle, main step is included：(1) magnetic nano-particle is modified and functionalization；(2) magnetic enrichment is carried out to the microorganism in food in liquid environment；(3) genome separation, purifying are carried out to the bacterium being enriched to；(4) bacterial genomes isolated and purified are carried out with the detection of nucleic acids of PCR-based.The program can be efficiently enriched with to the pathogenic bacteria in food in a short time, and be isolated and purified from bacterium is enriched to high-quality genomic templates, carry out detection of nucleic acids.It is quicker relative to traditional method, substantial amounts of human and material resources are saved, the pathogenic bacteria of low concentration in food can effectively be detected, testing result is also more reliable, it is easy to the promotion and application in fields such as production, life, scientific researches.
Description
Technical field
The invention belongs to technical field of biological, the functional magnetic nanometer of more particularly to a kind of efficiently concentrating microorganism
Particle and preparation and application.
Background technology
With social progress, food security is increasingly subject to people's attention, and especially the pathogenic bacteria in food is to people
Health generate serious threat.Detection method main at present is all to increase bacterium, flat-plate bacterial colony morphologic observation by culture medium
To be detected to microorganism, this method is relatively reliable, but also has this clearly disadvantageous.Culture medium increases bacterium, single bacterium colony and divided
And identification generally requires the time of more than 5 days, this can not meet the high circulation in present market and ageing demand, nothing
Infection of the method to food pathogenic is effectively prevented.Meanwhile, the detection based on colonial morphology is even more not very reliable, it is also desirable to specially
The personage of industry operates.Therefore, develop a kind of scheme that can carry out quickly, reliably detecting to food pathogenic and seem particularly urgent
Cut.Increasing bacterium generally requires the time for a long time, and is enriched with then opposite, it is only necessary to effectively collected the pathogen in certain system
In.A variety of enrichment modes to bacterium are occurred in that now, mainly by based on antibody, aptamers, antibiotic, change
Learn the mode that group etc. and bacterium can specifically bind, but these methods also all there are problems that, such as antibody price compared with
It is high and single bacterium can only be recognized, while being inconvenient to store and transporting；The nanometer magnetic bead of antibiotic such as vancomycin modification
Can be because the dispersiveness that the property that modification changes magnetic bead surfaces is allowed in a liquid weakens, under the capture rate of bacterium
Drop；Chemical group such as amino is combined often by charge effect to the electronegative bacterium in surface, and which results in nothing
Method efficiently separates the genome of purifying capture bacterium, and the detection of nucleic acid aspect can not subsequently be carried out to the bacterium captured by causing.
These defects resulted in currently used for enrichment magnetic bead can not real life, production in effectively be applied.Therefore open
Send that a kind of property is stable, easily stored, transport, wide spectrum, high efficiency can be carried out to bacterium and be enriched with, and not influence follow-up to bacterium
The functionalized nano magnetic bead that genome carries out detection of nucleic acids has important social effect and economic value.
The content of the invention
In order to overcome the shortcoming and deficiency of prior art, primary and foremost purpose of the invention is to provide a kind of efficiently concentrating micro- life
The preparation method of the functionalized magnetic nano particle of thing.
Another object of the present invention is to provide the functionalized magnetic nano particle obtained by above-mentioned preparation method.The work(
Magnetic nano-particle, which can be changed, to carry out efficiently concentrating and detection of nucleic acids to the pathogenic bacteria in food.
Another object of the present invention is to provide it is a kind of based on above-mentioned functions magnetic nano-particle to micro- life in food
The method that thing carries out efficiently concentrating and detection of nucleic acids.
The purpose of the present invention is achieved through the following technical solutions：A kind of functional magnetic nanoparticle of efficiently concentrating microorganism
The preparation method of son, comprises the following steps：
Vancomycin is modified onto amino magnetic bead, then uses one end activated carboxylic by amino, carboxyl dehydration condensation
Polyethylene glycol (PEG) remaining amino sites on magnetic bead are closed；Obtain the functionalization of energy efficiently concentrating microorganism
Magnetic nano-particle.
Described amino magnetic bead is the ferroferric oxide magnetic nanospheres purchased from Tianjin Bei Sile companies, surface group:-
NH2, particle diameter is 100~200nm, and concentration is 5mg/mL, is scattered in PBS；Ferroferric oxide magnetic nanospheres, its
English full name is Fe3O4Magnetic nanoparticles, are abbreviated as Fe3O4MNPs acute pyogenic infection of finger tip in MNPs, the present invention
Fe3O4MNPs；
Described PBS is 1 × PBS, and pH value is 7.4~7.6；The mother liquor of described PBS is
20 × PBS, pH value is 7.4~7.6, purchased from Sangon Biotech (Shanghai) Co., Ltd.；
The PEG of described one end activated carboxylic is preferably that its molecular weight is 2000~6000；More preferably 5000；
The PEG of described one end activated carboxylic is more preferably mono methoxy polyethylene glycol succinimdyl carbonate (mPEG-
SC), molecular weight is 5kDa, purchased from process engineering research institute of the Chinese Academy of Sciences；
Described amino, the reaction step and system of carboxyl dehydration condensation are as follows：
1) amino magnetic bead is taken, magnetic enrichment is carried out, 30mM/pH6 MES buffer solution for cleaning three times is added and is resuspended in 30mM/
In pH6 MES buffer solutions；
2) vancomycin hydrochloride, n-hydroxysuccinimide (NHS), 1- (3- dimethylamino-propyls) -3- ethyls are weighed
Carbodiimide hydrochloride (EDCHCl)；
3) by step 1) in amino magnetic bead and step 2) in medicine be uniformly mixed and added into 30mM/pH6 MES buffering
Liquid, obtains mixed liquor, reaction；
4) after the completion of reacting, Magneto separate, 30mM/pH6 MES buffer solution for cleaning three times are carried out to magnetic bead, and be resuspended in
In 30mM/pH6MES buffer solutions, obtain modifying the nanometer magnetic bead of vancomycin；
Described 30mM/pH6 MES buffer solutions are made by the steps：Take 30mmol/L MES, be dissolved in from
In sub- water, it is 6.0 to adjust pH value, obtains 30mM/pH6 MES buffer solutions；Wherein, MES is 2- (N- morpholines) ethyl sulfonic acid, is purchased from
Sigma-Aldrich (Shanghai) trade Co., Ltd；
Step 1) described in amino magnetic bead, particle diameter be 100~200nm, concentration is 5mg/mL, is scattered in PBS
In, with step 2) described in vancomycin hydrochloride volume mass than be preferably (400~425) mL：3g；
Step 2) described in vancomycin hydrochloride, n-hydroxysuccinimide (NHS) and 1- (3- dimethylaminos third
Base) mass ratioes of -3- ethyl-carbodiimide hydrochlorides (EDCHCl) is preferably 3:(5~7):(7~9)；
Step 3) described in mixed liquor in the concentration of vancomycin hydrochloride be preferably 3~6g/mL；
Step 3) described in reaction condition be preferably 20~30 DEG C, 170~190rpm reactions 5h~7h；
Step 4) described in be resuspended in 30mM/pH6MES buffer solutions, wherein, the volumes of MES buffer solutions and step 3) in
The volume ratio of described mixed liquor is preferably 1：1；
The reaction step and system of described closing is as follows：The nanometer magnetic bead 5mL of above-mentioned vancomycin modification is taken, is weighed
The PEG of 0.025g one end activated carboxylic, is well mixed, reaction；After the completion of reaction, Magneto separate is carried out to magnetic bead, 30mM/pH6's
MES buffer solution for cleaning three times, and being resuspended in 5mL 30mM/pH6MES buffer solutions, obtains vancomycin and the dual modifications of PEG
Nanometer magnetic bead, can efficiently concentrating microorganism functionalized magnetic nano particle；
The condition of described reaction is preferably 20~30 DEG C, 170~190rpm reactions 11h~13h；
A kind of functionalized magnetic nano particle of efficiently concentrating microorganism, is obtained by above-mentioned preparation method；
It is a kind of that efficiently concentrating and detection of nucleic acids are carried out to the microorganism in food based on above-mentioned functions magnetic nano-particle
Method, specifically include following steps：
(1) magnetic enrichment is carried out to the microorganism in food in liquid environment
Take the bacterial solution of finite concentration gradient to be distributed in LB culture mediums, above-mentioned functions magnetic Nano is added thereto
Particle, concussion reaction carries out magnetic enrichment, then carries out Magneto separate, be finally re-dispersed into the nano-particle for capturing bacterium
In 30mM/pH6 MES buffer solutions, clean 3 times, collect nano-particle/bacterial complex；
(2) genome separation, purifying are carried out to the bacterium being enriched to
The bacterium being enriched in the nano-particle/bacterial complex collected by enzymatic isolation method to step (1) cracks, so
Magnetic bead and cell pyrolysis liquid are separated by Magneto separate afterwards, then the bacterial genomes in cell pyrolysis liquid are isolated and purified；
(3) bacterial genomes isolated and purified are carried out with the detection of nucleic acids of PCR-based
The bacterial genomes isolated and purified in primer pair step (2) by optimizing design for tested bacteria are carried out
PCR is expanded, and obtains length-specific PCR primer, and carry out gel electrophoresis and ultraviolet photographic gel development experiment；
Described bacterium is preferably Listeria monocytogenes (Listeria monocytogenes)；
The bacterial solution of finite concentration gradient described in step (1) is to carry out incubated overnight in 10mL LB culture mediums
Bacterium, OD600 is 1, and concentration is about 1 × 108Cfu/mL, takes the above-mentioned bacterium solution 10000rpm of 1mL, 4 DEG C of centrifugation 2min, collects heavy
Form sediment, in the buffer solution for being resuspended in 1mL 30mM MES/0.1M NaCl pH6, and carry out 10 times of gradient dilutions, obtain 1 ×
106Cfu/mL to 1 × 100Each 1mL of cfu/mL dilution factor bacterium solutions；
Bacterial solution, LB culture mediums, above-mentioned functions magnetic nano-particle described in step (1) and again scattered
The volume ratio of MES buffer solutions is preferably 1:9:1:1；
Addition above-mentioned functions magnetic nano-particle described in step (1), first carries out Magneto separate to it, only adds solid
Composition；
The condition of concussion reaction described in step (1) is preferably 20~30 DEG C, 190~210rpm reactions, 25~35min；
The step of enzymatic isolation method described in step (2) is：The magnetic bead that the collection of 1mL functionalized magnetic nano particles is obtained/
Bacterial complex carries out Magneto separate, and 110 μ L TE buffer solutions are added thereto, is well mixed, adds 70 μ L lysozymes, 37 DEG C
React 1h；Reaction adds 20 μ L Proteinase Ks thereto after terminating, and is well mixed；
The condition of cracking described in step (2) is preferably 56 DEG C of cracking reaction 30min；
Bacterial genomes described in step (2) are isolated and purified to carry out after Magneto separate, and aqueous phase is transferred into 1.5mL
In centrifuge tube, the absolute ethyl alcohol of 220 μ L precoolings is added, concussion is mixed, brief centrifugation；Then examination is extracted with bacterial genomes DNA
Agent box is isolated and purified, and finally gives the μ L of bacterial genomes 50 of purifying；
The sequence of primer described in step (3) is：
PCR sense primers：5′-AGGAATGACTAATCAAGACAAT-3′；
PCR anti-sense primers：5′-GGAAGGTCTTGTAGGTTCATTA-3′；
The system and response procedures of PCR amplifications described in step (3) are as follows：
PCR system：
| PCR system | 50μL | Mother liquid concentration | Final concentration |
| PCR sense primers | 1μL | 10μM | 0.2μM |
| PCR anti-sense primers | 1μL | 10μM | 0.2μM |
| 10×PCR buffer | 5μL | - | - |
| dNTP | 4μL | - | - |
| Template | 2μL | 200ng/μL | 80ng/μL |
| RTaq enzymes | 0.2μL | - | - |
| Water | 37μL | - | - |
PCR response procedures：
The present invention mechanism be：Based on the natural efficient affinity interaction to bacteria cell wall of vancomycin, modified
Onto magnetic Nano microsphere, using the superparamagnetic property of magnetic Nano microsphere, in the presence of externally-applied magnetic field, with it in food
Pathogenic microorganisms carry out wide spectrum, the enrichment of orientation, then improve with the PEG of one end activated carboxylic the table of functionalized nano magnetic bead
Surface properties, greatly increase its dispersiveness under liquid system, the bioaccumulation efficiency to low concentration bacterium are improved, while changing it
Surface electrical behavior, allow to from the enrichment with magnetic bead to bacterium in effectively extract it is high-quality nucleic acid-templated, carry out nucleic acid
Molecule is diagnosed (see Fig. 1).
The present invention has the following advantages and effect relative to prior art：
(1) it is quicker relative to the method for increasing bacterium based on culture medium with the method for magnetic enrichment, operate also more convenient, section
Plenty of time and manpower are saved.
(2) have in a liquid with the functionalization magnetic bead of the dual modifications of PEG of vancomycin and one end activated carboxylic more preferable
Dispersiveness, its capture rate to bacterium is stronger, while the wide spectrum enrichment to bacterium can be realized, rather than is only confined to certain
Bacterium, and the bacterium arrived using the enrichment with magnetic bead are planted, high-quality bacterium also can effectively be extracted by conventional enzymatic isolation method
Genome, for follow-up detection of nucleic acids.
(3) the relatively conventional bacterium colony Psychology analysis method of the method for nucleic acid analysis of PCR-based is more quick, sensitivity
Higher, the diagnosis from nucleic acid aspect is also more definite.
(4) probe sequence designed in this programme is relative to other same PCR for being directed to Listeria monocytogenes Hly genes
Primer has higher sensitivity and specificity, it is ensured that effective detection and really is carried out to a small amount of pathogenic bacteria in food
Examine.
Brief description of the drawings
Fig. 1 is the side for carrying out efficiently concentrating and detection of nucleic acids to the microorganism in food based on functionalized magnetic nano particle
Method schematic diagram.
Fig. 2 is the transmission electron microscope photo and nanoparticle after the vancomycin and PEG that embodiment 1 is obtained are modified amino magnetic bead
Sublist face Zeta potential testing result figure.
Fig. 3 is that the functionalization magnetic bead that embodiment 2 is obtained carries out the clustering phenomena photo after magnetic enrichment to Listeria monocytogenes
And electromicroscopic photograph figure.
Fig. 4 is the agarose gel electrophoresis experimental data knot detected by this programme method to the bacterium of various concentrations
Fruit is schemed；Wherein, 1E5,1E4,1E3,1E2,1E1,1E0,0.1 represent that bacterial concentration is 1 × 10 respectively5cfu/mL、1×104cfu/
mL、1×103cfu/mL、1×102cfu/mL、1×101cfu/mL、1×100cfu/mL、0.1cfu/mL；NC is the LB of sterilizing
Culture medium, is negative control；
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
Following experimental methods are conventional method unless otherwise instructed, used experiment material unless otherwise instructed,
Easily it can be obtained from commercial company.
The formula of described LB culture mediums is tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, adjusts pH extremely
7.0,121 DEG C of autoclaving 20min；Solid LB media need to only add 15~20g/L of agar before sterilization.
Embodiment 1 carries out functionalization by vancomycin and PEG to amino magnetic bead
1. taking 4.14mL amino magnetic beads, magnetic enrichment is carried out on Magneto separate frame, 30mM/pH6 MES buffer solution for cleaning is added
Three times and it is resuspended in 1mL 30mM/pH6 MES buffer solutions.
2. vancomycin hydrochloride 0.03g is weighed on electronic balance, n-hydroxysuccinimide (NHS) 0.06g, 1-
(above medicine is purchased from Shanghai Sigma- to (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDCHCl) 0.08g
Aldrich)；
3. by step 1) in amino magnetic bead and step 2) in medicine add in 15mL centrifuge tube, and add 30mM/
PH6 MES buffer solutions, which complement to reaction system in 10mL, shaking table, to react, 25 DEG C of temperature, rotating speed 180rpm, time 6h.
4. after the completion of reaction, Magneto separate, 30mM/pH6 MES buffer solution for cleaning three times are carried out to magnetic bead, and be resuspended in
In 10mL 30mM/pH6MES buffer solutions, obtain modifying the nanometer magnetic bead product of vancomycin；
5. taking the nanometer magnetic bead 5mL of vancomycin in the modification that step 4 obtains, the mono- methoxies of 0.025g are weighed in electronic balance
Base polyethylene glycol succinimdyl carbonate, is well mixed in 15mL centrifuge tubes, is reacted in shaking table, and temperature is 25 DEG C of temperature,
Rotating speed 180rpm, time 12h.After the completion of reaction, to magnetic bead carry out Magneto separate, 30mM/pH6 MES buffer solution for cleaning three times, and
It is resuspended in 5mL 30mM/pH6MES buffer solutions, obtains the nanometer magnetic bead product of vancomycin and the dual modifications of PEG, Ji Nenggao
Imitate the functionalized magnetic nano particle of enriched microorganism.
The nanometer magnetic bead and step 5 of vancomycin are obtained in the modification obtained to step 4 vancomycin and PEG is dual repaiies
The nanometer magnetic bead of decorations, is taken pictures by transmission electron microscope respectively, and Fig. 2 (A) and Fig. 2 (B) are seen respectively；Fig. 2 (C) is nanoparticle sublist
Face Zeta potential testing result figure.
The magnetic bead of the functionalization of embodiment 2 carries out magnetic enrichment to the microorganism in food
Microorganism in food is pathogenic bacteria bacterium：Listeria monocytogenes (Listeria monocytogenes) (bacterial strain
Number be CMCC54007, purchased from Guangzhou institute of microbiology), in 10mL LB culture mediums carry out incubated overnight bacterium, OD600
For 1, concentration is about 1 × 108Cfu/mL, takes the above-mentioned bacterium solution 10000rpm of 1mL, 4 DEG C of centrifugation 2min, collects precipitation, be resuspended in 1mL
In 30mM MES/0.1M NaCl pH6 buffer solution, and 10 times of gradient dilutions are carried out, obtain 1 × 106Cfu/mL to 1 ×
100Each 1mL of cfu/mL dilution factor bacterium solutions.
Above-mentioned 1 × 10 is added in 15mL centrifuge tubes respectively6Cfu/mL to 1 × 100Cfu/mL bacterium solution 1mL, are added thereto
Then the LB culture mediums of sterilizing, supplement volume to 10mL is separately added into process double labeling prepared in embodiment 1 thereto
Magnetic bead 1mL (first carry out Magneto separate, only add solid constituent), react in shaking table, temperature is 25 DEG C of temperature, rotating speed 200rpm,
Time 30min.After the completion of reaction, Magneto separate is carried out, 30mM/pH6 MES buffer solution for cleaning 3 times is resuspended in 1mL 30mM/
In pH6MES buffer solutions, magnetic bead/bacterial complex is obtained.
Listeria monocytogenes are carried out the aggregation after magnetic enrichment and showed by the functionalized magnetic nano particle obtained with embodiment 1
As photo and electromicroscopic photograph figure, as shown in Figure 3；As can be seen from Figure 3：The functionalized magnetic nano particle of acquisition can increase to single
Listeria reaches good concentration effect.
Embodiment 3 is detected by this programme platform to the bacterium of various concentrations
The magnetic bead obtained in embodiment 2/bacterial complex is subjected to Magneto separate, 110 μ L TE buffer solutions are added thereto,
It is well mixed, add and reacted in 70 μ L lysozymes (being purchased from Tiangeng biochemical technology Co., Ltd), water-bath, temperature is 37 DEG C,
Time is 1h.Reaction adds 20 μ L Proteinase Ks (being purchased from Tiangeng biochemical technology Co., Ltd) thereto after terminating, be well mixed,
Reacted in water-bath, temperature is 56 DEG C, the time is to carry out Magneto separate after the completion of 30min, reaction, by aqueous phase be transferred to 1.5mL from
In heart pipe, the absolute ethyl alcohol of 220 μ L precoolings is added, concussion is mixed, brief centrifugation.
Above-mentioned product is separated with bacterial genomes DNA extraction kit (being purchased from Tiangeng biochemical technology Co., Ltd)
Purifying, finally gives the μ L of bacterial genomes 50 of purifying.
The bacterial genomes isolated and purified by the primer pair of design enter performing PCR amplification, obtain length-specific PCR primer,
And carry out gel electrophoresis and ultraviolet photographic gel development experiment；
The sequence of described primer is as follows：
PCR sense primers：5′-AGGAATGACTAATCAAGACAAT-3′；
PCR anti-sense primers：5′-GGAAGGTCTTGTAGGTTCATTA-3′；
The system and response procedures of described PCR amplifications are as follows：
PCR system：
| PCR system | 50μL | Mother liquid concentration | Final concentration |
| PCR sense primers | 1μL | 10μM | 0.2μM |
| PCR anti-sense primers | 1μL | 10μM | 0.2μM |
| 10×PCR buffer | 5μL | - | - |
| dNTP | 4μL | - | - |
| Template | 2μL | 200ng/μL | 80ng/μL |
| RTaq enzymes | 0.2μL | - | - |
| Water | 37μL | - | - |
PCR response procedures：
The target fragment that PCR amplifications are obtained is Hly target fragments, and its sequence is：Total length 325bp
AGGAATGACTAATCAAGACAATAAAATCGTTGTAAAAAATGCTACTAAATCGAATGTTAACAACGCAGTAAATACAT
TAGTGGAAAGATGGAATGAAAAATATGCTCAAGCTTATCCGAATGTAAGTGCAAAAATTGATTATGATGACGAAATG
GCTTACAGTGAATCACAATTAATTGCAAAATTTGGTACTGCATTTAAAGCTGTAAATAATAGCTTGAATGTAAACTT
CGGCGCAATCAGTGAAGGGAAAATGCAAGAAGAAGTCATTAGTTTTAAACAAATTTACTATAACGTGAATGTTAATG
AACCTACAAGACCTTCC。
The PCR primer for taking 5 μ L above-mentioned, carries out 1% (g/mL) agarose and coagulates gel electrophoresis and ultraviolet photographic gel development experiment.
As shown in figure 4, as a result showing：With preferable detection sensitivity, bacterial concentration reaches 1 × 103More than cfu/mL,
What can accurately be detected arrives.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (6)
1. a kind of preparation method of the functionalized magnetic nano particle of efficiently concentrating microorganism, it is characterised in that including following step
Suddenly：
Vancomycin is modified onto amino magnetic bead by amino, carboxyl dehydration condensation, then gathering with one end activated carboxylic
Ethylene glycol is closed to remaining amino sites on magnetic bead；Obtain the functional magnetic nanoparticle of energy efficiently concentrating microorganism
Son；
Described amino magnetic bead is ferroferric oxide magnetic nanospheres, surface group:-NH2, particle diameter is 100~200nm, concentration
For 5mg/mL, it is scattered in PBS；
Described amino, the reaction step and system of carboxyl dehydration condensation are as follows：
1) amino magnetic bead is taken, magnetic enrichment is carried out, 30mM/pH6 MES buffer solution for cleaning three times is added and is resuspended in 30mM/pH6's
In MES buffer solutions；
2) vancomycin hydrochloride, n-hydroxysuccinimide, 1- (3- dimethylamino-propyls) -3- ethyl carbodiimide salt are weighed
Hydrochlorate；
3) by step 1) in amino magnetic bead and step 2) in medicine be uniformly mixed and added into 30mM/pH6 MES buffer solutions,
Mixed liquor is obtained, is reacted；
4) after the completion of reacting, Magneto separate, 30mM/pH6 MES buffer solution for cleaning three times are carried out to magnetic bead, and be resuspended in 30mM/
In pH6MES buffer solutions, obtain modifying the nanometer magnetic bead of vancomycin；
Step 1) described in amino magnetic bead and step 2) described in vancomycin hydrochloride volume mass ratio for (400~
425)mL：3g；
Step 2) described in vancomycin hydrochloride, n-hydroxysuccinimide and 1- (3- dimethylamino-propyls) -3- ethyls
The mass ratio of carbodiimide hydrochloride is 3:(5~7):(7~9)；
Step 3) described in mixed liquor in vancomycin hydrochloride concentration be 3~6g/mL.
2. preparation method according to claim 1, it is characterised in that：
The polyethylene glycol of described one end activated carboxylic is that its molecular weight is 2000~6000.
3. preparation method according to claim 1, it is characterised in that：The reaction step and system of described closing is as follows：
The nanometer magnetic bead 5mL of above-mentioned vancomycin modification is taken, the PEG of 0.025g one end activated carboxylic is weighed, is well mixed, reaction；Instead
After the completion of answering, Magneto separate, 30mM/pH6 MES buffer solution for cleaning three times are carried out to magnetic bead, and be resuspended in 5mL 30mM/pH6MES
In buffer solution, obtain the nanometer magnetic bead of vancomycin and the dual modifications of PEG, can the functional magnetic of efficiently concentrating microorganism receive
Rice corpuscles；
The condition of described reaction is 20~30 DEG C, 170~190rpm reactions 11h~13h.
4. a kind of functionalized magnetic nano particle of efficiently concentrating microorganism, it is characterised in that pass through any one of claims 1 to 3
Described preparation method is obtained.
5. a kind of functionalized magnetic nano particle based on described in claim 4 in food microorganism carry out efficiently concentrating and
The method of detection of nucleic acids, it is characterised in that specifically include following steps：
(1) magnetic enrichment is carried out to the microorganism in food in liquid environment
Take the bacterial solution of finite concentration gradient to be distributed in LB culture mediums, above-mentioned functions magnetic nano particle is added thereto
Son, concussion reaction carries out magnetic enrichment, then carries out Magneto separate, be finally re-dispersed into the nano-particle for capturing bacterium
In 30mM/pH6 MES buffer solutions, clean 3 times, collect nano-particle/bacterial complex；
(2) genome separation, purifying are carried out to the bacterium being enriched to
The bacterium being enriched in the nano-particle/bacterial complex collected by enzymatic isolation method to step (1) cracks, Ran Houtong
Cross Magneto separate to separate magnetic bead and cell pyrolysis liquid, then the bacterial genomes in cell pyrolysis liquid are isolated and purified；
(3) bacterial genomes isolated and purified are carried out with the detection of nucleic acids of PCR-based
The bacterial genomes isolated and purified in primer pair step (2) by optimizing design for tested bacteria enter performing PCR
Amplification, obtains length-specific PCR primer, and carry out gel electrophoresis and ultraviolet photographic gel development experiment；
Described bacterium is Listeria monocytogenes (Listeria monocytogenes)；
The sequence of primer described in step (3) is：
PCR sense primers：5′-AGGAATGACTAATCAAGACAAT-3′；
PCR anti-sense primers：5′-GGAAGGTCTTGTAGGTTCATTA-3′.
6. method according to claim 5, it is characterised in that：
Bacterial solution, LB culture mediums, above-mentioned functions magnetic nano-particle and again scattered MES described in step (1) are delayed
The volume ratio of fliud flushing is 1:9:1:1；
The condition of concussion reaction described in step (1) is 20~30 DEG C, 190~210rpm reactions, 25~35min；
The condition of cracking described in step (2) is 56 DEG C of cracking reaction 30min.
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