CN104313130A - Functionalized magnetic nanoparticles for efficiently enriching microorganisms, preparation and applications thereof - Google Patents
Functionalized magnetic nanoparticles for efficiently enriching microorganisms, preparation and applications thereof Download PDFInfo
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Abstract
The present invention discloses functionalized magnetic nanoparticles for efficiently enriching microorganisms, preparation and applications thereof. The method for carrying out efficient enrichment and nucleic acid detection on microorganisms in food mainly comprises: (1) modifying and functionalizing magnetic nanoparticles; (2) carrying out magnetic enrichment on microorganisms in food in a liquid environment; (3) carrying out genome isolation and purification on the enriched bacteria; and (4) carrying out PCR-based nucleic acid detection on the isolated and purified bacterial genome. With the technical scheme, the pathogenic bacteria in the food can be efficiently enriched within a short time, and the high quality genome template is isolated and purified from the enriched bacteria and then is subjected to nucleic acid detection. Compared with the traditional method, the method of the present invention has characteristics of rapidity, manpower saving, material resource saving, efficient detection of the low concentration pathogenic bacteria in the food, and reliable detection result, and is easily generalized and applied in the fields of production, life, scientific research and the like.
Description
Technical field
The invention belongs to technical field of biological, particularly the functionalized magnetic nano particle of a kind of efficiently concentrating microorganism and preparation and application.
Background technology
Along with social progress, food safety is day by day subject to people and payes attention to, and the health of the pathogenic bacteria especially in food to people creates serious threat.Detection method main be at present all increase bacterium by substratum, flat-plate bacterial colony morphologic observation detects microorganism, this method is relatively reliable, but also has this significantly not enough.Substratum increases bacterium, single bacterium colony divides and identify the time often needing more than 5 days, and this cannot meet present market high circulation and ageing demand, effectively cannot prevent the infection of food pathogenic.Meanwhile, the detection based on colonial morphology is very unreliable especially, needs the personage of specialty to operate yet.Therefore, develop a kind ofly to carry out fast food pathogenic, the reliable scheme detected seems particularly urgent.Increasing bacterium often needs the time for a long time, and enrichment is then contrary, and only the pathogenic bacteria in certain system carries out effective concentrated by needs.There is the multiple enrichment mode to bacterium now, mainly pass through based on the mode with bacterium energy specific binding such as antibody, aptamers, microbiotic, chemical group, but these methods also all also exist some problems, such as antibody price is higher and can only identify single bacterium, is inconvenient to store and transport simultaneously; The nanometer magnetic bead that microbiotic such as vancomycin is modified because the dispersiveness that the character that modification changes magnetic bead surfaces makes it in a liquid weakens, can decline to the capture rate of bacterium; Chemical group is such as amino to be combined often by the electronegative bacterium of charge effect effects on surface, and which results in and effectively separation and purification cannot catch the genome of bacterium, causes the follow-up detection cannot carrying out nucleic acid aspect to the bacterium captured.These defects all result in effectively cannot be applied for the magnetic bead of enrichment at present in real life, production.Therefore develop a kind of stable in properties, be easy to store, transport, wide spectrum, high-level efficiency enrichment can be carried out to bacterium, and not affect and follow-uply to the functionalized nano magnetic bead that bacterial genomes carries out detection of nucleic acids, there is important social effect and economic worth.
Summary of the invention
In order to overcome the shortcoming of prior art with not enough, primary and foremost purpose of the present invention is the preparation method of the functionalized magnetic nano particle providing a kind of efficiently concentrating microorganism.
Another object of the present invention is to provide the functionalized magnetic nano particle obtained by above-mentioned preparation method.This functionalized magnetic nano particle can carry out efficiently concentrating and detection of nucleic acids to the pathogenic bacteria in food.
Another object of the present invention is to provide a kind of method of based on above-mentioned functions magnetic nano-particle, the microorganism in food being carried out to efficiently concentrating and detection of nucleic acids.
Object of the present invention is achieved through the following technical solutions: the preparation method of the functionalized magnetic nano particle of a kind of efficiently concentrating microorganism, comprises the steps:
Vancomycin is modified on amino magnetic bead by amino, carboxyl dehydration condensation, then uses the polyoxyethylene glycol of one end activated carboxylic (PEG) to close amino sites remaining on magnetic bead; Namely the functionalized magnetic nano particle of energy efficiently concentrating microorganism is obtained.
Described amino magnetic bead is the ferroferric oxide magnetic nanospheres purchased from Tianjin Bei Sile company, surface group :-NH
2, particle diameter is 100 ~ 200nm, and concentration is 5mg/mL, is scattered in PBS damping fluid; Ferroferric oxide magnetic nanospheres, its English full name is Fe
3o
4magnetic nanoparticles, is abbreviated as Fe
3o
4mNPs, MNPs acute pyogenic infection of finger tip Fe in the present invention
3o
4mNPs;
Described PBS damping fluid is 1 × PBS damping fluid, and pH value is 7.4 ~ 7.6; The mother liquor of described PBS damping fluid is 20 × PBS damping fluid, and pH value is 7.4 ~ 7.6, purchased from Sangon Biotech (Shanghai) Co., Ltd.;
It is 2000 ~ 6000 that the PEG of described one end activated carboxylic is preferably its molecular weight; Be more preferably 5000;
The PEG of described one end activated carboxylic is more preferably mono methoxy polyethylene glycol succinimdyl carbonate (mPEG-SC), and molecular weight is 5kDa, purchased from process engineering institute of the Chinese Academy of Sciences;
Reactions steps and the system of described amino, carboxyl dehydration condensation are as follows:
1) get amino magnetic bead, carry out magnetic enrichment, add the MES buffer solution for cleaning three times of 30mM/pH6 and be resuspended in the MES damping fluid of 30mM/pH6;
2) vancomycin hydrochloride is taken, N-hydroxy-succinamide (NHS), 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl);
3) by step 1) in amino magnetic bead and step 2) in medicine Homogeneous phase mixing and add the MES damping fluid of 30mM/pH6, obtain mixed solution, reaction;
4) after having reacted, Magneto separate is carried out to magnetic bead, the MES buffer solution for cleaning of 30,mM/,pH6 three times, and be resuspended in 30mM/pH6MES damping fluid, obtain the nanometer magnetic bead modifying vancomycin;
The MES damping fluid of described 30mM/pH6 is prepared as follows: the MES getting 30mmol/L, is dissolved in deionized water, and adjust pH is 6.0, obtains the MES damping fluid of 30mM/pH6; Wherein, MES and 2-(N-morpholine) ethyl sulfonic acid, purchased from Sigma-Aldrich (Shanghai) trade Co., Ltd;
Step 1) described in amino magnetic bead, particle diameter is 100 ~ 200nm, and concentration is 5mg/mL, is scattered in PBS damping fluid, with step 2) described in vancomycin hydrochloride volume mass than be preferably (400 ~ 425) mL:3g;
Step 2) described in the mass ratio of vancomycin hydrochloride, N-hydroxy-succinamide (NHS) and 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) be preferably 3:(5 ~ 7): (7 ~ 9);
Step 3) described in mixed solution in the concentration of vancomycin hydrochloride be preferably 3 ~ 6g/mL;
Step 3) described in the condition optimization of reaction be 20 ~ 30 DEG C, 170 ~ 190rpm reacts 5h ~ 7h;
Step 4) described in be resuspended in 30mM/pH6MES damping fluid, wherein, the volume of MES damping fluid and step 3) described in the volume ratio of mixed solution be preferably 1:1;
The described reactions steps closed and system as follows: get the nanometer magnetic bead 5mL that above-mentioned vancomycin is modified, take the PEG of 0.025g one end activated carboxylic, mix, reaction; After having reacted, Magneto separate is carried out to magnetic bead, the MES buffer solution for cleaning of 30,mM/,pH6 three times, and be resuspended in 5mL 30mM/pH6MES damping fluid, obtain the nanometer magnetic bead of vancomycin and the dual modification of PEG, can the functionalized magnetic nano particle of efficiently concentrating microorganism;
The condition optimization of described reaction is 20 ~ 30 DEG C, 170 ~ 190rpm reacts 11h ~ 13h;
A functionalized magnetic nano particle for efficiently concentrating microorganism, is obtained by above-mentioned preparation method;
Based on above-mentioned functions magnetic nano-particle, the microorganism in food is carried out to a method for efficiently concentrating and detection of nucleic acids, specifically comprise the steps:
(1) in liquid environment, magnetic enrichment is carried out to the microorganism in food
The Bacteria liquid getting finite concentration gradient is distributed in LB substratum, add above-mentioned functions magnetic nano-particle wherein, concussion reaction, carry out magnetic enrichment, then Magneto separate is carried out, finally the nanoparticle capturing bacterium is re-dispersed in the MES damping fluid of 30mM/pH6, cleans 3 times, collect nanoparticle/bacterial complex;
(2) gene element is carried out from, purifying to the bacterium be enriched to
The bacterium be enriched in the nanoparticle/bacterial complex collected step (1) by enzymolysis process carries out cracking, then by Magneto separate by magnetic bead and cell pyrolysis liquid separately, then carries out separation and purification to the bacterial genomes in cell pyrolysis liquid;
(3) bacterial genomes of separation and purification is carried out to the detection of nucleic acids of PCR-based
Carry out pcr amplification by the bacterial genomes of the middle separation and purification of primer pair step (2) being optimized design for tested bacteria, obtain length-specific PCR primer, and carry out gel electrophoresis and ultraviolet photograph glue experiment;
Described bacterium is preferably Listeria monocytogenes (Listeria monocytogenes);
The Bacteria liquid of the finite concentration gradient described in step (1) is, in 10mL LB substratum, carry out the bacterium of incubated overnight, OD600 is 1, and concentration is about 1 × 10
8cfu/mL, gets 1mL above-mentioned bacterium liquid 10000rpm, 4 DEG C of centrifugal 2min, collecting precipitation, is resuspended in the damping fluid of 1mL 30mM MES/0.1M NaCl pH6, and carries out 10 times of gradient dilutions, obtain 1 × 10
6cfu/mL to 1 × 10
0the each 1mL of cfu/mL extent of dilution bacterium liquid;
Bacteria liquid described in step (1), LB substratum, above-mentioned functions magnetic nano-particle and the volume ratio of MES damping fluid of again disperseing are preferably 1:9:1:1;
Add above-mentioned functions magnetic nano-particle described in step (1), first Magneto separate is carried out to it, only add solids component;
The condition optimization of the concussion reaction described in step (1) is 20 ~ 30 DEG C, 190 ~ 210rpm reacts 25 ~ 35min;
The step of the enzymolysis process described in step (2) is: 1mL functionalized magnetic nano particle is collected the magnetic bead/bacterial complex obtained and carries out Magneto separate, add 110 μ L TE damping fluids wherein, mix, then add 70 μ L N,O-Diacetylmuramidases, 37 DEG C of reaction 1h; Add 20 μ L Proteinase Ks wherein after reaction terminates, mix;
The condition optimization of the cracking described in step (2) is 56 DEG C of scission reaction 30min;
Bacterial genomes described in step (2) carries out separation and purification for after carrying out Magneto separate, is transferred to by aqueous phase in 1.5mL centrifuge tube, adds the dehydrated alcohol of 220 μ L precoolings, concussion mixing, brief centrifugation; Then carry out separation and purification by bacterial genomes DNA extraction kit, finally obtain the bacterial genomes 50 μ L of purifying;
The sequence of the primer described in step (3) is:
PCR upstream primer: 5 '-AGGAATGACTAATCAAGACAAT-3 ';
PCR downstream primer: 5 '-GGAAGGTCTTGTAGGTTCATTA-3 ';
System and the response procedures of the pcr amplification described in step (3) are as follows:
PCR system:
| PCR system | 50μL | Mother liquid concentration | Final concentration |
| PCR upstream primer | 1μL | 10μM | 0.2μM |
| PCR downstream primer | 1μL | 10μM | 0.2μM |
| 10×PCR?buffer | 5μL | - | - |
| dNTP | 4μL | - | - |
| Template | 2μL | 200ng/μL | 80ng/μL |
| RTaq enzyme | 0.2μL | - | - |
| Water | 37μL | - | - |
PCR response procedures:
Mechanism of the present invention is: based on the efficient affinity interaction to bacteria cell wall that vancomycin is natural, modified on magnetic Nano microsphere, utilize the superparamagnetic character of magnetic Nano microsphere, under the effect of externally-applied magnetic field, with it, wide spectrum is carried out to the pathogenic microorganism in food, directed enrichment, the surface properties of functionalized nano magnetic bead is improved again with the PEG of one end activated carboxylic, its dispersiveness under liquid system is increased greatly, improve the bioaccumulation efficiency to lower concentration bacterium, change its surface electrical behavior simultaneously, allow to from this enrichment with magnetic bead to bacterium effectively extract high-quality nucleic acid-templated, carry out nucleic acid molecule diagnosis (see Fig. 1).
The present invention, relative to prior art, has following advantage and effect:
(1) more quick relative to the method increasing bacterium based on substratum by the method for magnetic enrichment, operate also more convenient, save plenty of time and manpower.
(2) with the functionalization magnetic bead of the dual modification of PEG of vancomycin and one end activated carboxylic, there is better dispersiveness in a liquid, it is stronger to the capture rate of bacterium, the wide spectrum enrichment to bacterium can be realized simultaneously, instead of be only confined to certain bacterium, and use the bacterium that this enrichment with magnetic bead arrives, also high-quality bacterial genomes can be effectively extracted, for follow-up detection of nucleic acids by the enzymolysis process of routine.
(3) the bacterium colony Psychology analysis method that the method for nucleic acid analysis of PCR-based is relatively traditional is more quick, and sensitivity is also higher, also more definite from the diagnosis of nucleic acid aspect.
(4) designed in this programme probe sequence has higher sensitivity and specificity for the PCR primer of Listeria monocytogenes Hly gene equally relative to other, ensure that and effectively to detect pathogenic bacteria a small amount of in food and make a definite diagnosis.
Accompanying drawing explanation
Fig. 1 is the Method And Principle figure based on functionalized magnetic nano particle, the microorganism in food being carried out to efficiently concentrating and detection of nucleic acids.
Fig. 2 be embodiment 1 obtain vancomycin and PEG to amino magnetic bead modify after transmission electron microscope photo and nanoparticle surface Zeta potential detected result figure.
Fig. 3 is that the functionalization magnetic bead that embodiment 2 obtains carries out the clustering phenomena photo after magnetic enrichment and electromicroscopic photograph figure to Listeria monocytogenes.
Fig. 4 is the agarose gel electrophoresis experimental data figure detected by the bacterium of this programme method to different concns; Wherein, 1E5,1E4,1E3,1E2,1E1,1E0,0.1 represent that bacterial concentration is 1 × 10 respectively
5cfu/mL, 1 × 10
4cfu/mL, 1 × 10
3cfu/mL, 1 × 10
2cfu/mL, 1 × 10
1cfu/mL, 1 × 10
0cfu/mL, 0.1cfu/mL; NC is the LB substratum of sterilizing, is negative control;
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Following experimental technique if no special instructions, is ordinary method, and the experiment material used if no special instructions, all can easily obtain from commercial company.
The formula of described LB substratum is Tryptones 10g/L, yeast extract 5g/L, NaCl 10g/L, adjusts pH to 7.0,121 DEG C of autoclaving 20min; Solid LB media only need add agar 15 ~ 20g/L before sterilization.
Embodiment 1 carries out functionalization by vancomycin and PEG to amino magnetic bead
1. get 4.14mL amino magnetic bead, Magneto separate frame carries out magnetic enrichment, add the MES buffer solution for cleaning three times of 30mM/pH6 and be resuspended in the MES damping fluid of 1mL 30mM/pH6.
2. on electronic balance, take vancomycin hydrochloride 0.03g, N-hydroxy-succinamide (NHS) 0.06g, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) 0.08g (above medicine is all purchased from Shanghai Sigma-Aldrich company);
3. by step 1) in amino magnetic bead and step 2) in medicine add in the centrifuge tube of 15mL, and reaction system is complemented to 10mL by the MES damping fluid adding 30mM/pH6, reacts in shaking table, temperature 25 DEG C, rotating speed 180rpm, time 6h.
4. after having reacted, Magneto separate is carried out to magnetic bead, the MES buffer solution for cleaning of 30,mM/,pH6 three times, and be resuspended in 10mL 30mM/pH6MES damping fluid, obtain the nanometer magnetic bead product modifying vancomycin;
5. get the nanometer magnetic bead 5mL of vancomycin in modification that step 4 obtains, take 0.025g mono methoxy polyethylene glycol succinimdyl carbonate at electronic balance, mix in 15mL centrifuge tube, react in shaking table, temperature is temperature 25 DEG C, rotating speed 180rpm, time 12h.After having reacted, Magneto separate is carried out to magnetic bead, the MES buffer solution for cleaning of 30,mM/,pH6 three times, and be resuspended in 5mL 30mM/pH6MES damping fluid, obtain the nanometer magnetic bead product of vancomycin and the dual modification of PEG, can the functionalized magnetic nano particle of efficiently concentrating microorganism.
The vancomycin that in the modification obtain step 4, the nanometer magnetic bead of vancomycin and step 5 obtain and the nanometer magnetic bead of the dual modification of PEG, take pictures respectively by transmission electron microscope, see Fig. 2 (A) and Fig. 2 (B) respectively; Fig. 2 (C) is nanoparticle surface Zeta potential detected result figure.
The magnetic bead of embodiment 2 functionalization carries out magnetic enrichment to the microorganism in food
Microorganism in food is pathogenic bacterium bacterium: (bacterial strain number is CMCC54007 to Listeria monocytogenes (Listeria monocytogenes), purchased from Guangzhou institute of microbiology), the bacterium of incubated overnight is carried out in 10mL LB substratum, OD600 is 1, and concentration is about 1 × 10
8cfu/mL, gets 1mL above-mentioned bacterium liquid 10000rpm, 4 DEG C of centrifugal 2min, collecting precipitation, is resuspended in the damping fluid of 1mL 30mM MES/0.1M NaCl pH6, and carries out 10 times of gradient dilutions, obtain 1 × 10
6cfu/mL to 1 × 10
0the each 1mL of cfu/mL extent of dilution bacterium liquid.
Above-mentioned 1 × 10 is added respectively in 15mL centrifuge tube
6cfu/mL to 1 × 10
0cfu/mL bacterium liquid 1mL, add the LB substratum of sterilizing wherein, supplement volume to 10mL, then add the magnetic bead 1mL through double-tagging prepared in embodiment 1 wherein respectively and (first carry out Magneto separate, only add solids component), react in shaking table, temperature is temperature 25 DEG C, rotating speed 200rpm, time 30min.After having reacted, carry out Magneto separate, the MES buffer solution for cleaning of 30,mM/,pH6 3 times, be resuspended in 1mL 30mM/pH6MES damping fluid, obtain magnetic bead/bacterial complex.
With the functionalized magnetic nano particle that embodiment 1 obtains, the clustering phenomena photo after magnetic enrichment and electromicroscopic photograph figure are carried out to Listeria monocytogenes, as shown in Figure 3; As can be seen from Figure 3: the functionalized magnetic nano particle of acquisition can reach good concentration effect to Listeria monocytogenes.
Embodiment 3 is detected by the bacterium of this programme platform to different concns
Magnetic bead/the bacterial complex obtained in embodiment 2 is carried out Magneto separate, adds 110 μ L TE damping fluids wherein, mix, add 70 μ L N,O-Diacetylmuramidases (purchased from Tian Gen biochemical technology company limited) again, react in water-bath, temperature is 37 DEG C, and the time is 1h.20 μ L Proteinase Ks (purchased from Tian Gen biochemical technology company limited) are added wherein after reaction terminates, mix, react in water-bath, temperature is 56 DEG C, and the time is 30min, carries out Magneto separate after having reacted, aqueous phase is transferred in 1.5mL centrifuge tube, add the dehydrated alcohol of 220 μ L precoolings, concussion mixing, brief centrifugation.
Above-mentioned product bacterial genomes DNA extraction kit (purchased from Tian Gen biochemical technology company limited) is carried out separation and purification, finally obtains the bacterial genomes 50 μ L of purifying.
Carry out pcr amplification by the bacterial genomes of the primer pair separation and purification of design, obtain length-specific PCR primer, and carry out gel electrophoresis and ultraviolet photograph glue experiment;
The sequence of described primer is as follows:
PCR upstream primer: 5 '-AGGAATGACTAATCAAGACAAT-3 ';
PCR downstream primer: 5 '-GGAAGGTCTTGTAGGTTCATTA-3 ';
System and the response procedures of described pcr amplification are as follows:
PCR system:
| PCR system | 50μL | Mother liquid concentration | Final concentration |
| PCR upstream primer | 1μL | 10μM | 0.2μM |
| PCR downstream primer | 1μL | 10μM | 0.2μM |
| 10×PCR?buffer | 5μL | - | - |
| dNTP | 4μL | - | - |
| Template | 2μL | 200ng/μL | 80ng/μL |
| RTaq enzyme | 0.2μL | - | - |
| Water | 37μL | - | - |
PCR response procedures:
The target fragment that pcr amplification obtains is Hly target fragment, and its sequence is: total length 325bp
AGGAATGACTAATCAAGACAATAAAATCGTTGTAAAAAATGCTACTAAATCGAATGTTAACAACGCAGTAAATACATTAGTGGAAAGATGGAATGAAAAATATGCTCAAGCTTATCCGAATGTAAGTGCAAAAATTGATTATGATGACGAAATGGCTTACAGTGAATCACAATTAATTGCAAAATTTGGTACTGCATTTAAAGCTGTAAATAATAGCTTGAATGTAAACTTCGGCGCAATCAGTGAAGGGAAAATGCAAGAAGAAGTCATTAGTTTTAAACAAATTTACTATAACGTGAATGTTAATGAACCTACAAGACCTTCC。
Get the PCR primer that 5 μ L are above-mentioned, carry out 1% (g/mL) agarose and coagulate gel electrophoresis and ultraviolet photograph glue experiment.
As shown in Figure 4, result shows: have good detection sensitivity, bacterial concentration reaches 1 × 10
3more than cfu/mL, that all can accurately detect arrives.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. a preparation method for the functionalized magnetic nano particle of efficiently concentrating microorganism, is characterized in that comprising the steps:
Vancomycin is modified on amino magnetic bead by amino, carboxyl dehydration condensation, then with the polyoxyethylene glycol of one end activated carboxylic, amino sites remaining on magnetic bead is closed; Namely the functionalized magnetic nano particle of energy efficiently concentrating microorganism is obtained.
2. preparation method according to claim 1, is characterized in that: described amino magnetic bead is ferroferric oxide magnetic nanospheres, surface group :-NH
2, particle diameter is 100 ~ 200nm, and concentration is 5mg/mL, is scattered in PBS damping fluid;
The polyoxyethylene glycol of described one end activated carboxylic for its molecular weight be 2000 ~ 6000.
3. preparation method according to claim 1, is characterized in that: reactions steps and the system of described amino, carboxyl dehydration condensation are as follows:
1) get amino magnetic bead, carry out magnetic enrichment, add the MES buffer solution for cleaning three times of 30mM/pH6 and be resuspended in the MES damping fluid of 30mM/pH6;
2) vancomycin hydrochloride is taken, N-hydroxy-succinamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride;
3) by step 1) in amino magnetic bead and step 2) in medicine Homogeneous phase mixing and add the MES damping fluid of 30mM/pH6, obtain mixed solution, reaction;
4) after having reacted, Magneto separate is carried out to magnetic bead, the MES buffer solution for cleaning of 30,mM/,pH6 three times, and be resuspended in 30mM/pH6MES damping fluid, obtain the nanometer magnetic bead modifying vancomycin.
4. preparation method according to claim 3, is characterized in that: step 1) described in amino magnetic bead and step 2) described in the volume mass of vancomycin hydrochloride than for (400 ~ 425) mL:3g;
Step 2) described in the mass ratio of vancomycin hydrochloride, N-hydroxy-succinamide and 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride be 3:(5 ~ 7): (7 ~ 9);
Step 3) described in mixed solution in the concentration of vancomycin hydrochloride be 3 ~ 6g/mL.
5. preparation method according to claim 1, is characterized in that: the described reactions steps closed and system as follows: get the nanometer magnetic bead 5mL that above-mentioned vancomycin is modified, take the PEG of 0.025g one end activated carboxylic, mix, reaction; After having reacted, Magneto separate is carried out to magnetic bead, the MES buffer solution for cleaning of 30,mM/,pH6 three times, and be resuspended in 5mL 30mM/pH6MES damping fluid, obtain the nanometer magnetic bead of vancomycin and the dual modification of PEG, can the functionalized magnetic nano particle of efficiently concentrating microorganism;
The condition of described reaction is 20 ~ 30 DEG C, 170 ~ 190rpm reacts 11h ~ 13h.
6. a functionalized magnetic nano particle for efficiently concentrating microorganism, is obtained by the preparation method described in any one of Claims 1 to 5.
7. based on functionalized magnetic nano particle according to claim 6, microorganism in food is carried out to a method for efficiently concentrating and detection of nucleic acids, it is characterized in that specifically comprising the steps:
(1) in liquid environment, magnetic enrichment is carried out to the microorganism in food
The Bacteria liquid getting finite concentration gradient is distributed in LB substratum, add above-mentioned functions magnetic nano-particle wherein, concussion reaction, carry out magnetic enrichment, then Magneto separate is carried out, finally the nanoparticle capturing bacterium is re-dispersed in the MES damping fluid of 30mM/pH6, cleans 3 times, collect nanoparticle/bacterial complex;
(2) gene element is carried out from, purifying to the bacterium be enriched to
The bacterium be enriched in the nanoparticle/bacterial complex collected step (1) by enzymolysis process carries out cracking, then by Magneto separate by magnetic bead and cell pyrolysis liquid separately, then carries out separation and purification to the bacterial genomes in cell pyrolysis liquid;
(3) bacterial genomes of separation and purification is carried out to the detection of nucleic acids of PCR-based
Carry out pcr amplification by the bacterial genomes of the middle separation and purification of primer pair step (2) being optimized design for tested bacteria, obtain length-specific PCR primer, and carry out gel electrophoresis and ultraviolet photograph glue experiment.
8. method according to claim 7, is characterized in that: described bacterium is Listeria monocytogenes (Listeria monocytogenes).
9. method according to claim 8, is characterized in that:
The sequence of the primer described in step (3) is:
PCR upstream primer: 5 '-AGGAATGACTAATCAAGACAAT-3 ';
PCR downstream primer: 5 '-GGAAGGTCTTGTAGGTTCATTA-3 '.
10. method according to claim 7, is characterized in that:
Bacteria liquid described in step (1), LB substratum, above-mentioned functions magnetic nano-particle and the volume ratio of MES damping fluid of again disperseing are 1:9:1:1;
The condition of the concussion reaction described in step (1) is 20 ~ 30 DEG C, 190 ~ 210rpm reacts 25 ~ 35min;
The condition of the cracking described in step (2) is 56 DEG C of scission reaction 30min.
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