CN107988207A - Extracted from complex samples and purify the kit and method of salmonella DNA - Google Patents
Extracted from complex samples and purify the kit and method of salmonella DNA Download PDFInfo
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- CN107988207A CN107988207A CN201711431673.2A CN201711431673A CN107988207A CN 107988207 A CN107988207 A CN 107988207A CN 201711431673 A CN201711431673 A CN 201711431673A CN 107988207 A CN107988207 A CN 107988207A
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- 101710099276 Probable metalloendopeptidase Proteins 0.000 claims abstract description 36
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 30
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 30
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 30
- 239000000243 solution Substances 0.000 claims abstract description 23
- 238000002955 isolation Methods 0.000 claims abstract description 14
- 238000010521 absorption reaction Methods 0.000 claims abstract description 11
- 239000003480 eluent Substances 0.000 claims abstract description 10
- 239000006166 lysate Substances 0.000 claims abstract description 10
- 239000012898 sample dilution Substances 0.000 claims abstract description 9
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- 238000000746 purification Methods 0.000 claims abstract description 8
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 9
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 7
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 7
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 7
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 101710083735 Gene 13 protein Proteins 0.000 claims description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 3
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000005336 cracking Methods 0.000 claims description 3
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 239000003761 preservation solution Substances 0.000 claims description 3
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- 102000016943 Muramidase Human genes 0.000 claims description 2
- 108010014251 Muramidase Proteins 0.000 claims description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 2
- 229960000274 lysozyme Drugs 0.000 claims description 2
- 235000010335 lysozyme Nutrition 0.000 claims description 2
- 239000004325 lysozyme Substances 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 239000000047 product Substances 0.000 claims 1
- 238000007400 DNA extraction Methods 0.000 abstract description 2
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- 239000002689 soil Substances 0.000 description 2
- SCYULBFZEHDVBN-UHFFFAOYSA-N 1,1-Dichloroethane Chemical class CC(Cl)Cl SCYULBFZEHDVBN-UHFFFAOYSA-N 0.000 description 1
- 208000031295 Animal disease Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
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- -1 Ethyl sulfonic acid monohydrate Chemical class 0.000 description 1
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- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
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- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229940099352 cholate Drugs 0.000 description 1
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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- 229940116269 uric acid Drugs 0.000 description 1
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- 206010048282 zoonosis Diseases 0.000 description 1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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Abstract
The invention discloses a kind of kit and method extracted from complex samples and purify salmonella DNA, belong to biology field.The present invention extracts from complex samples and purifies the kit of salmonella DNA, including：1）It has been coupled the carboxylated nanometer magnetic bead CPBA MNPs of gp13 albumen；2）Sample dilution；3）Sample lysate；4）Nucleic acid cleaning solution；5）DNA eluents 6）Nucleic acid absorption magnetic bead.The present invention then purifies salmonella using the carboxylated nanometer magnetic bead CPBA MNPs specific binding salmonellas for being coupled gp13 albumen, and the paramagnetic particle method of salmonella after purification then is extracted DNA.Isolation and purification culture of the whole process without salmonella, directly carries out DNA extractions；Simplify experimentation, greatly save experimental period.
Description
Technical field
It is more particularly to a kind of to be extracted from complex samples and purify salmonella the present invention relates to biology field
The kit and method of DNA.
Background technology
Salmonella（Salmonella）It is Gram-negative enteric bacterium, a kind of common Zoonosis pathogen, not only
Cause various Animal diseases, and it is related with a variety of diseases of the mankind.Salmonella can be divided into Salmonella enteritidis and Bang Geersha
Door 2 kinds of Salmonella, Salmonella enteritidis can be divided into 6 subspecies.
Salmonella bacteriophage P22 is a kind of virus using salmonella as host, and host specificity is very strong.Gp13 is located at
Bacteriophage P22 long tailfibers end, its C-terminal can identify host strain surface receptor, trigger phage adsorption to salmonella surface
Process, be bacteriophage P22 identification receptors key protein.
Complex samples refer to food, excrement, soil, vomitus equal samples, these samples generally all containing suppress downstream PCR or
The inhibitor of person NGS detections, such as complicated polysaccharide, cholate, lipid and uric acid etc..Tradition is from complex samples（Food, excrement, soil
Earth, vomitus etc.）The method of middle extraction salmonella DNA needs first to separate from sample and purify culture bacterial strain, then from purifying
Nucleic acid is extracted in good bacterial strain, process is more complicated, and the time used is longer.
The content of the invention
In order to make up for the deficiencies of the prior art, the existing procedure that salmonella DNA is extracted from complex samples is solved
Complicated, the problem of required time is long, the present invention provides a kind of reagent extracted from complex samples and purify salmonella DNA
Box and method.
The technical scheme is that：
A kind of kit extracted from complex samples and purify salmonella DNA, it is characterised in that including：
1）It has been coupled the carboxylated nanometer magnetic bead CPBA-MNPs of gp13 albumen；
2）Sample dilution；
3）Sample lysate；
4）Nucleic acid cleaning solution；
5）DNA eluents
6）Nucleic acid absorption magnetic bead.
Preferably, step 1）The preparation method of the middle carboxyl nanometer magnetic bead CPBA-MNPs for being coupled gp13 albumen
For：
a）Using the salmonella expression system of gp13 albumen, solubility expression gp13 albumen simultaneously purifies；
b）Activated hydroxyl groups nanometer magnetic bead：
c）Gp13 albumen is coupled with hydroxylating nanometer magnetic bead.
Further, step c）The detailed process that gp13 albumen is coupled with hydroxylating nanometer magnetic bead is：
Using the borate solution containing Tween-20 as coupling buffer, hydroxylating nano magnetic is cleaned with the coupling buffer
Pearl；
The hydroxylating nanometer magnetic bead, gp13 albumen after purification and coupling buffer are mixed, 2-4 is coupled at 20-30 DEG C
Hour；Magnetic Isolation removes supernatant, adds PBST solution and magnetic bead, 20-30 DEG C of reaction 30-90min closing magnetic bead surfaces are resuspended
Unreacted activated hydroxyl groups group；
Magnetic Isolation supernatant, after PBS or preservation solution washing, is placed in and preserves in solution, 4 DEG C of preservations.
Preferably, the pH of the coupling buffer is 7.5-8.5.It is furthermore preferred that the pH of the coupling buffer
For 8.5.
Preferably, the composition of the Sample dilution is：NaCl 137mM, KCl 2.7mM, Na2HPO4
4.3mM, KH2PO41.4mM, 0.05%Tween-20；
The composition of the sample lysate is：NaCl 0.05mM, EDTA 5mM, the Tris-HCl 20mM of PH=7.5, bacteriolyze
Enzyme 3mg/ml.
Preferably, the composition of the nucleic acid cleaning solution is：NaCl 0.05mM, EDTA 0.05mM, PH=7.5
Tris-HCl 20mM, absolute ethyl alcohol 70%.
Preferably, the composition of the DNA eluents is：EDTA 5mM, the Tris-HCl 10mM of PH=8.8.
The method extracted using the kit from complex samples and purify salmonella DNA, including step：
1）Complex samples are taken, add the sample diluting liquid, after mixing, centrifuging and taking supernatant；
2）Add the carboxylated nanometer magnetic bead CPBA-MNPs for being coupled gp13 albumen, shaken cultivation 10- at 36.5-37.5 DEG C
20min；
3）Magnetic Isolation combines the magnetic bead of salmonella, removes liquid, and the magnetic bead magnetic bead for combining salmonella uses sample
Dilution cleans；
4）Sample lysate is added into the magnetic bead for combine salmonella, 15-20min is incubated at 36.5-37.5 DEG C, is cracked
Thalline；
5）After cracking, it is cooled to room temperature, adds nucleic acid absorption magnetic bead, room temperature mixes；
6）The magnetic bead for being adsorbed with nucleic acid is cleaned using nucleic acid cleaning solution；
7）DNA eluents are added into the magnetic bead for be adsorbed with nucleic acid, are incubated at 36.5-37.5 DEG C, then Magnetic Isolation, supernatant
Preserved at -20 DEG C.
Preferably, step 1）In, 8-10 mL sample diluting liquids are added in 1g complex samples；Step 2）Middle coupling
The carboxylated nanometer magnetic bead CPBA-MNPs of gp13 albumen and step 1）The volume ratio of supernatant is 1:0.6-1.5.
Preferably, step 4）In, the volume ratio of the magnetic bead and sample lysate that combine salmonella is 1:3-
5；Step 5）In, the volume ratio of nucleic acid absorption magnetic bead and the magnetic bead for combining salmonella is 2.5-3.5:2；Step 7）In, DNA
The volume ratio of eluent and nucleic acid absorption magnetic bead is 1:5-7.
Beneficial effects of the present invention are：
The present invention specifically binds salmonella then to sramana using the carboxylated nanometer magnetic bead CPBA-MNPs for being coupled gp13
Salmonella is purified, and the paramagnetic particle method of salmonella after purification then is extracted DNA.Separation of the whole process without salmonella
Purifying culture, directly carries out DNA extractions；Simplify experimentation, greatly save experimental period.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is attached drawing needed in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, without having to pay creative labor, may be used also
To obtain other attached drawings according to these attached drawings.
Fig. 1 is the electrophoretogram of gp13 albumen after purification；
Fig. 2 is that the soak time of hydroxylating nanometer magnetic bead influences situation map to gp13 albumen couplings amount；
Fig. 3 is that the pH of coupling buffer influences situation map to gp13 albumen couplings amount；
Fig. 4 is influence situation map of the coupling time to gp13 albumen coupling amounts；
Fig. 5 is the design sketch of the method for the present invention PCR nucleic acid extractions.
Embodiment
Embodiment 1
A kind of kit extracted from complex samples and purify salmonella DNA, including：
1）It has been coupled the carboxylated nanometer magnetic bead CPBA-MNPs of gp13 albumen；
2）Sample dilution（Reagent A）；
3）Sample lysate（Reagent B）；
4）Nucleic acid cleaning solution（Reagent C）；
5）DNA eluents（Reagent D）
6）Nucleic acid absorption magnetic bead；
Wherein,
The composition of reagent A is：NaCl 137mM, KCl 2.7mM, Na2HPO4 4.3mM, KH2PO41.4mM, 0.05%
Tween-20。
The composition of reagent B is：NaCl 0.05mM, EDTA 5mM, the Tris-HCl 20mM of PH=7.5, lysozyme
3mg/ml。
The composition of reagent C is：NaCl 0.05mM, EDTA 0.05mM, the Tris-HCl 20mM of PH=7.5, anhydrous second
Alcohol 70%.
The composition of reagent D is：EDTA 5mM, the Tris-HCl 10mM of PH=8.8.
The preparation method for being coupled the carboxyl nanometer magnetic bead CPBA-MNPs of gp13 albumen is：
a）Using the salmonella expression system of gp13 albumen, solubility expression gp13 albumen simultaneously purifies；Gp13 eggs after purification
White protein electrophoresis figure is as shown in Figure 1.
b）Activated hydroxyl groups nanometer magnetic bead；
Carboxylated nanometer magnetic bead uses the nanometer magnetic bead of commercialization（Suzhou castor biomedical engineering Co., Ltd MagCOOH）,
After mixing magnetic bead, 100 uL Mag COOH magnetic beads are taken into 1.5 mL centrifuge tubes, and Magnetic Isolation removes supernatant, with 200 uL
MEST solution（100 mM MES (2-（N- morpholines）Ethyl sulfonic acid monohydrate）, pH 5.0,0.05% Tween 20）Carry out magnetic
Separating, washing 2 times, then removes supernatant；
It is rapidly added 100 μ L EDC of Fresh（Dichloroethanes）Solution（10 mg/mL, are disperseed with above-mentioned MEST solution
Agent）With 100 μ L NHS（10 mg/mL, make dispersant with above-mentioned MEST solution）Solution is into the centrifuge tube equipped with magnetic bead, whirlpool
Mixing makes magnetic bead fully suspend, and 25 DEG C of 30 min of activation, are kept the suspended state of magnetic bead, carried out using vertical mixed instrument during this period
It is reverse to mix；After above-mentioned steps, the carboxyl of magnetic bead surfaces is activated, can with the bio-ligand with primary amino radical into
Row covalent coupling.
The soak time of hydroxylating nanometer magnetic bead influences than more significant albumen coupling amount, as shown in Fig. 2, when magnetic bead activates
Between most preferably 30min, soak time is long or too short can all influence gp13 albumen coupling amounts.
c）Gp13 albumen is coupled with hydroxylating nanometer magnetic bead.
Step c）The detailed process that gp13 albumen is coupled with hydroxylating nanometer magnetic bead is：
A. Magnetic Isolation removes supernatant, with Tween-20 of the borate solution of 1mmol/L containing 0.05g/dL（Abbreviation BST）Make
For coupling buffer, first magnetic bead being cleaned with BST 2 times, adding the gp13 of appropriate BST and 150ug after purification, volume is settled to
300uL；Wherein, the pH of coupling buffer is 7.5-8.5, is most preferably 8.0.The pH of coupling buffer is to gp13 albumen coupling amounts
Influence significantly, as shown in figure 3, when coupling buffer pH is 8.0, gp13 albumen couplings amount is maximum.
B.25 DEG C 2 h of coupling, reverse mixing is carried out using vertical mixed instrument；
Coupling time also has considerable influence to gp13 albumen coupling amounts, as shown in figure 4, when coupling time is 2 small, gp13 albumen is even
Connection amount is maximum.
C. centrifuge tube is placed in Magnetic Isolation on magnetic separation rack and removes supernatant, adds 300 uL PBST solution（pH
7.2, and contain 1%BSA）Magnetic bead is resuspended, 25 DEG C of reaction 1 h closing unreacted activated carboxyl groups of magnetic bead surfaces, keep during this period
The suspended state of magnetic bead.
D. centrifuge tube is placed in Magnetic Isolation on magnetic separator and removes supernatant, every time with 200 uL PBS solutions（pH
7.2）Or after preservation solution washs 3 times, it is resuspended in preserving solution（Containing 0.02g/dLNaN3BST-BSA）In, obtain even
Join the carboxylated nanometer magnetic bead CPBA-MNPs of gp13 albumen, be stored in 4 DEG C.
The method extracted using kit from complex samples and purify salmonella DNA, including step：
1）1g complex samples are taken, add 9mL reagent As in the centrifuge tube of 50mL, after fully mixing, centrifugation, takes 200 μ L of supernatant liquid
In the centrifuge tube of 1.5mL；
2）Add the carboxylated nanometer magnetic bead CPBA-MNPs that 200 μ L have been coupled gp13 albumen, 37 DEG C, 100rpm shaken cultivations
15min；
3）The magnetic bead that magnetic frame Magnetic Isolation combines salmonella is placed in, removes liquid, combines the magnetic bead magnetic of salmonella
Pearl is cleaned at least 3 times using 400 μ L reagent As；
4）800 μ L reagent B are added into the magnetic bead for combine salmonella, 15-20min is incubated at 37 DEG C, crack thalline；
5）After cracking, centrifuge tube is taken out from incubation equipment, is cooled to room temperature, adds nucleic acid absorption magnetic bead（It is purchased from
BioMag article No.s：BMQ300) 300 μ L, room temperature mix；Centrifuge tube is placed in magnetic separation on magnetic frame, waste liquid is removed and inhales
Net tube cover and tube bottom residual night；
6）The magnetic bead for being adsorbed with nucleic acid is cleaned using 1mL reagent Cs, magnetic separation, the tube cover that exhausts and tube bottom residual night；Repeat above-mentioned step
Suddenly twice, uncap at room temperature and dry；
7）50 μ L reagent Ds are added into the magnetic bead for be adsorbed with nucleic acid, it is anti-terminate in water-bath incubate after magnetic separation, draw supernatant
Liquid preserves in new EP pipes at -20 DEG C.
Fig. 5 is the design sketch that nucleic acid extraction is carried out using the method for the present invention, and as shown in Figure 5, the present invention can be carried effectively
The nucleic acid of salmonella is taken, and extraction process need not isolate and purify sample, can effectively save from various complex samples
Extract the time of nucleic acid.
Claims (10)
- A kind of 1. kit extracted from complex samples and purify salmonella DNA, it is characterised in that including：1）It has been coupled the carboxylated nanometer magnetic bead CPBA-MNPs of gp13 albumen；2）Sample dilution；3）Sample lysate；4）Nucleic acid cleaning solution；5）DNA eluents6）Nucleic acid absorption magnetic bead.
- 2. extracted as claimed in claim 1 from complex samples and purify the kit of salmonella DNA, it is characterised in that step Rapid 1）The preparation method of the middle carboxyl nanometer magnetic bead CPBA-MNPs for being coupled gp13 albumen is：a）Using the salmonella expression system of gp13 albumen, solubility expression gp13 albumen simultaneously purifies；b）Activated hydroxyl groups nanometer magnetic bead：c）Gp13 albumen is coupled with hydroxylating nanometer magnetic bead.
- 3. extracted as claimed in claim 2 from complex samples and purify the kit of salmonella DNA, it is characterised in that step Rapid c）The detailed process that gp13 albumen is coupled with hydroxylating nanometer magnetic bead is：Using the borate solution containing Tween-20 as coupling buffer, hydroxylating nano magnetic is cleaned with the coupling buffer Pearl；The hydroxylating nanometer magnetic bead, gp13 albumen after purification and coupling buffer are mixed, 2-4 is coupled at 20-30 DEG C Hour；Magnetic Isolation removes supernatant, adds PBST solution and magnetic bead, 20-30 DEG C of reaction 30-90min closing magnetic bead surfaces are resuspended Unreacted activated hydroxyl groups group；Magnetic Isolation supernatant, after PBS or preservation solution washing, is placed in and preserves in solution, 4 DEG C of preservations.
- 4. extracted as claimed in claim 3 from complex samples and purify the kit of salmonella DNA, it is characterised in that institute The pH for stating coupling buffer is 7.5-8.5.
- 5. extracted as claimed in claim 1 from complex samples and purify the kit of salmonella DNA, it is characterised in that institute The composition for stating Sample dilution is：The composition of the Sample dilution is：NaCl 137mM, KCl 2.7mM, Na2HPO4 4.3mM, KH2PO41.4mM, 0.05%Tween-20；The composition of the sample lysate is：NaCl 0.05mM, EDTA 5mM, the Tris-HCl 20mM of PH=7.5, lysozyme 3mg/ml.
- 6. extracted as claimed in claim 1 from complex samples and purify the kit of salmonella DNA, it is characterised in that institute The composition for stating nucleic acid cleaning solution is：NaCl 0.05mM, EDTA 0.05mM, the Tris-HCl 20mM of PH=7.5, anhydrous second Alcohol 70%.
- 7. extracted as claimed in claim 1 from complex samples and purify the kit of salmonella DNA, it is characterised in that institute The composition for stating DNA eluents is：EDTA 5mM, the Tris-HCl 10mM of PH=8.8.
- 8. the method extracted from complex samples using kit described in claim 1 and purified salmonella DNA, its feature exist In, including step：1）Complex samples are taken, add the sample diluting liquid, after mixing, centrifuging and taking supernatant；2）Add the carboxylated nanometer magnetic bead CPBA-MNPs for being coupled gp13 albumen, shaken cultivation 10- at 36.5-37.5 DEG C 20min；3）Magnetic Isolation combines the magnetic bead of salmonella, removes liquid, and the magnetic bead magnetic bead for combining salmonella uses sample Dilution cleans；4）Sample lysate is added into the magnetic bead for combine salmonella, 15-20min is incubated at 36.5-37.5 DEG C, is cracked Thalline；5）After cracking, it is cooled to room temperature, adds nucleic acid absorption magnetic bead, room temperature mixes；6）The magnetic bead for being adsorbed with nucleic acid is cleaned using nucleic acid cleaning solution；7）DNA eluents are added into the magnetic bead for be adsorbed with nucleic acid, are incubated at 36.5-37.5 DEG C, then Magnetic Isolation, supernatant Preserved at -20 DEG C.
- 9. the method extracted as claimed in claim 8 from complex samples and purify salmonella DNA, it is characterised in that：Step 1）In, 8-10 mL sample diluting liquids are added in 1g complex samples；Step 2）The middle carboxylated nanometer magnetic bead for being coupled gp13 albumen CPBA-MNPs and step 1）The volume ratio of supernatant is 1:0.6-1.5.
- 10. the method extracted as claimed in claim 8 from complex samples and purify salmonella DNA, it is characterised in that：Step 4）In, the volume ratio of the magnetic bead and sample lysate that combine salmonella is 1:3-5；Step 5）In, nucleic acid absorption magnetic bead with The volume ratio for combining the magnetic bead of salmonella is 2.5-3.5:2；Step 7）In, the body of DNA eluents and nucleic acid absorption magnetic bead Product is than being 1:5-7.
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| CN104651496A (en) * | 2015-01-19 | 2015-05-27 | 四川大学 | Method for rapidly detecting salmonella based on immunomagnetic bead-multiplex PCR |
| CN106093377A (en) * | 2016-08-12 | 2016-11-09 | 江苏福隆生物技术有限公司 | A kind of test kit for the quick coupling of albumen, antibody, enzyme and nucleic acid and preparation method thereof |
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| CN101921838A (en) * | 2010-06-11 | 2010-12-22 | 江南大学 | Method for detecting salmonella through PCR-DHPLC |
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