CN102121002A - Method for extracting bacterium genomic DNA by using magnetic nanoparticles - Google Patents
Method for extracting bacterium genomic DNA by using magnetic nanoparticles Download PDFInfo
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- CN102121002A CN102121002A CN201010602179XA CN201010602179A CN102121002A CN 102121002 A CN102121002 A CN 102121002A CN 201010602179X A CN201010602179X A CN 201010602179XA CN 201010602179 A CN201010602179 A CN 201010602179A CN 102121002 A CN102121002 A CN 102121002A
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Abstract
The invention discloses a method for extracting bacterium genomic DNA by using magnetic nanoparticles, belonging to the technical field of nanomaterials and molecular biology. The method comprises the steps of: preparing a magnetic nanoparticle aggregate, preparing a bacterium lysing solution, preparing and adding an absorption buffer solution, absorbing with a magnet, rinsing with 70 percent alcohol, and eluting with a TE buffer solution to obtain a bacterium genomic DNA solution. Compared with the traditional phenol/chloroform DNA extracting method, the method for extracting the bacterium genomic DNA by using magnetic nanoparticles has the advantages of short experiment time, simpleness in operation, high extraction efficiency, rapidness, convenience, sensitivity, high yield and the like; and the extracted bacterium genomic DNA can be completely used for subsequent experiments, such as DNA hybridization, PCR (Polymerase Chain Reaction), and the like, and provides the basis for the subsequent research and analysis.
Description
Technical field
The present invention relates to a kind of method that adopts magnetic nano-particle to extract bacterial genomes DNA, belong to nano material and technical field of molecular biology.
Background technology
Bacterium is distributed widely in nature, and wherein some pathogenic micro-organism can be invaded human body and animal, causes to infect even transmissible disease, all can constitute very big harm to human and animal's health.Edible by the food of these bacterial contaminations, to drink by the water of bacterial contamination be the main route of transmission of food origin disease.According to statistics, the annual actual food poisoning that takes place of China has 20-40 ten thousand people at least.In recent years, food poisoning increased year by year.The invasive organism that causes food poisoning is more, the most commonly enteritis vibrios, Salmonellas, Campylobacter and pathogenic colibacillus.
The method of traditional bacteriologic test needs biochemical cultivation, isolation identification, and the program complexity is loaded down with trivial details, the report assay roughly needs 4～7d, and is not only consuming time oversize, and detection sensitivity is low.So, set up the key problem that rapid and precise detection method is the pathogenic micro-organism Inspection Research always.Along with development of molecular biology, the polymerase chain reaction method (PCR) based on nucleic acid has been widely used in the testing of pathogenic micro-organism bacterium, compares with traditional method, and this detection method is quicker, more convenient, sensitiveer.
At present, the extracting method that is widely used in bacterial genomes DNA mainly is traditional separate nucleic acid method, exist to use deficiencies such as toxic reagent (phenol, chloroform), length consuming time, extraction efficiency are low, this can not reach modern detect to fast, accurately, sensitive, requirement easily.Therefore be necessary to invent the extracting method of a kind of new bacterial genomes DNA, magnetic micrometer, nanometer materials particle by inorganic or organic polymer finishing or parcel can overcome these deficiencies as the carrier of separate nucleic acid.
Summary of the invention
The extracting method that the purpose of this invention is to provide a kind of bacterial genomes DNA quick, accurate, easy and simple to handle is for DNA hybridization and polymerase chain reaction (PCR) and follow-up sequencing reaction provide approach.
Technical scheme of the present invention: a kind of method that adopts magnetic nano-particle to extract bacterial genomes DNA, comprise the preparation of magnetic nanometer particle congery, the preparation of bacterial lysate, the preparation of adsorption-buffering liquid and interpolation, magnet absorption, the TE buffer solution elution is used in ethanol rinsing with 70%, obtains the bacterial genomes dna solution;
(1) preparation of magnetic nanometer particle congery.
1. take by weighing 0.65g FERRIC CHLORIDE ANHYDROUS and 0.4g trisodium citrate, measure 20mL ethylene glycol, stir 20min, make it whole dissolvings;
2. take by weighing the 1.2g anhydrous sodium acetate and join in the above-mentioned reaction system, stir 10min, place the oil bath of tetrafluoroethylene reactor to be heated to 230 ℃, carry out magnetic agitation simultaneously, stop heating behind the reaction 10h, continue magnetic agitation and be cooled to room temperature;
3. reacted solution adds the dehydrated alcohol of 20mL, ultrasonic cleaning 10min, and the centrifugal 10min of 5000rpm abandons supernatant;
4. lower sediment adds the 20mL dehydrated alcohol, ultrasonic cleaning 10min, and the centrifugal 10min of 5000rpm abandons supernatant;
5. lower sediment adds the 20mL deionized water, ultrasonic cleaning 10min, and the centrifugal 10min of 5000rpm abandons supernatant;
6. lower sediment is suspended in the 10mL deionized water, gets magnetic nanometer particle congery, and room temperature preservation is standby.
(2) preparation of bacterial lysate:
For Gram-negative bacteria, get 1mL bacterium enrichment liquid in the 2mL centrifuge tube of sterilizing, the centrifugal 5min of 10000rpm outwells supernatant liquor, and in the 1mL bacterial lysate, vortex concussion 15s mixes 70 ℃ of water-bath 10min with resolution of precipitate;
Described bacterial lysate consists of: 50mM Tris-HCl, 10mM EDTA, 1% SDS, 5mg/mL Proteinase K, pH8.0;
Or for gram-positive microorganism, get 1mL bacterium enrichment liquid in the 2mL centrifuge tube of sterilizing, the centrifugal 5min of 10000rpm, outwell supernatant liquor, precipitation is dissolved in the 500 μ L buffer A, vortex concussion 15s mixes, behind 37 ℃ of water-bath 30min, add in the 500 μ L buffer B, vortex concussion 15s mixes 70 ℃ of water-bath 10min;
Described buffer A consists of: contain 50mM Tris-HCl, 10mM EDTA, 1.2% Triton, 20mg/mL N,O-Diacetylmuramidase, pH8.0;
Described buffer B consists of: contain 1% SDS, 5mg/mL Proteinase K, pH8.0.
(3) preparation of adsorption-buffering liquid and interpolation:
Preparation adsorption-buffering liquid PEG/NaCl(10%/6M) aqueous solution, wherein polyethylene glycol 6000 mass concentration 10%, NaCl concentration 6M;
In step (2) gained 1mL bacterial lysate, add the magnetic nanometer particle congery 10 μ L that prepare, add the adsorption-buffering liquid PEG/NaCl aqueous solution 500 μ L again, make the NaCl final concentration be controlled to be 2M, turn upside down for several times, mix, concussion reaction 10min, the magnetic nano-particle of acquisition absorption bacterial genomes DNA.
(4) magnet absorption: will be adsorbed with magnetic nano-particle and the solution separating of bacterial genomes DNA with magnet, and outwell supernatant liquor.
(5) with 70% ethanol rinsing: clean the magnetic nano-particle twice that is adsorbed with bacterial genomes DNA with 70% ethanol rinsing liquid, room temperature is placed 0.5h, makes the residual ethanol volatilization fully.
(6) use the TE buffer solution elution: add 50 μ LTE damping fluids, 10min is hatched in 65 ℃ of water-baths behind the mixing, and magnetic separates, and gets supernatant, is the bacterial genomes dna solution, is stored in-20 ℃, and is standby.
Described TE damping fluid is: the mixing solutions that contains 10mM Tris-HCl, 1mM EDTA, pH8.0.
Beneficial effect of the present invention: the present invention utilizes the synthetic magnetic nano-particle to extract the DNA of bacterial genomes, compare traditional phenol/chloroform DNA extraction method, whole experiment weak point consuming time, simple to operate, the extraction efficiency height, the bacterial genomes DNA that extracts can be used for subsequent experimental such as DNA hybridization and PCR fully, for later researching and analysing provides the foundation.
Description of drawings
The transmission electron microscope of Fig. 1 magnetic nano-particle (TEM) figure.
The agarose gel electrophoresis figure of the bacterial genomes DNA that Fig. 2 magnetic nano-particle extracts.
Embodiment
(1) preparation of magnetic nanometer particle congery:
①
Take by weighing 0.65g FERRIC CHLORIDE ANHYDROUS and 0.4g trisodium citrate, measure 20ml ethylene glycol, stir 20min, make it whole dissolvings;
2. take by weighing the 1.2g anhydrous sodium acetate and join in the above-mentioned reaction system, stir 10min, place the oil bath of tetrafluoroethylene reactor to be heated to 230 ℃, carry out magnetic agitation simultaneously, stop heating behind the reaction 10h, continue magnetic agitation and be cooled to room temperature;
3. reacted solution adds the dehydrated alcohol of 20ml, ultrasonic cleaning 10min, and the centrifugal 10min of 5000rpm abandons supernatant;
4. lower sediment adds the 20ml dehydrated alcohol, ultrasonic cleaning 10min, and the centrifugal 10min of 5000rpm abandons supernatant;
5. lower sediment adds the 20ml deionized water, ultrasonic cleaning 10min, and the centrifugal 10min of 5000rpm abandons supernatant;
6. lower sediment is dissolved in the 10ml deionized water, and room temperature preservation is standby.
(2) magnetic nano-particle extracts bacterial genomes DNA
1. the preparation of bacterial lysate:
For Gram-negative bacteria, get 1mL bacterium enrichment liquid in the 2mL centrifuge tube of sterilizing, the centrifugal 5min of 10000rpm outwells supernatant liquor, with resolution of precipitate (50mM Tris-HCl in the 1mL bacterial lysate, 10mM EDTA, 1%SDS, the 5mg/mL Proteinase K, pH8.0), vortex concussion 15s mixes 70 ℃ of water-bath 10min;
For gram-positive microorganism, get 1mL bacterium enrichment liquid in the 2mL centrifuge tube of sterilizing, the centrifugal 5min of 10000rpm, outwell supernatant liquor, precipitation is dissolved in 500 μ L damping fluid (50mM Tris-HCl, 10mM EDTA, 1.2% Triton, the 20mg/mL N,O-Diacetylmuramidase, pH8.0), vortex concussion 15s mixes, and behind 37 ℃ of water-bath 30min, adds 500 μ L solution (1%SDS, the 5mg/mL Proteinase K, pH8.0), vortex concussion 15s mixes 70 ℃ of water-bath 10min;
2. the preparation of adsorption-buffering liquid and interpolation: add the magnetic nano-particle prepare, 500 μ L adsorption-buffering liquid PEG/NaCl(10%/6M), making the NaCl final concentration is 2M, turns upside down for several times, mixes concussion reaction 10min;
3. magnet absorption: will be adsorbed with magnetic nano-particle and the solution separating of bacterial genomes DNA with magnet, and outwell supernatant liquor;
4. use rinsing liquid (70% ethanol) to clean magnetic nano-particle twice, room temperature is placed half an hour, makes the residual ethanol volatilization fully;
5. (pH8.0), 10min is hatched in 65 ℃ of water-baths behind the mixing for 10mM Tris-HCl, 1mM EDTA, and magnetic separates, and gets supernatant, is dna solution to add 50 μ L elution buffers.Be stored in-20 ℃, standby.
Claims (1)
1. method that adopts magnetic nano-particle to extract bacterial genomes DNA, it is characterized in that comprising the preparation of magnetic nanometer particle congery, the preparation of bacterial lysate, the preparation of adsorption-buffering liquid and interpolation, magnet absorption, the TE buffer solution elution is used in ethanol rinsing with 70%, obtains the bacterial genomes dna solution;
(1) preparation of magnetic nanometer particle congery:
1. take by weighing 0.65g FERRIC CHLORIDE ANHYDROUS and 0.4g trisodium citrate, measure 20mL ethylene glycol, stir 20min, make it whole dissolvings;
2. take by weighing the 1.2g anhydrous sodium acetate and join in the above-mentioned reaction system, stir 10min, place the oil bath of tetrafluoroethylene reactor to be heated to 230 ℃, carry out magnetic agitation simultaneously, stop heating behind the reaction 10h, continue magnetic agitation and be cooled to room temperature;
3. reacted solution adds the dehydrated alcohol of 20mL, ultrasonic cleaning 10min, and the centrifugal 10min of 5000rpm abandons supernatant;
4. lower sediment adds the 20mL dehydrated alcohol, ultrasonic cleaning 10min, and the centrifugal 10min of 5000rpm abandons supernatant;
5. lower sediment adds the 20mL deionized water, ultrasonic cleaning 10min, and the centrifugal 10min of 5000rpm abandons supernatant;
6. lower sediment is suspended in the 10mL deionized water, gets magnetic nanometer particle congery, and room temperature preservation is standby;
(2) preparation of bacterial lysate:
For Gram-negative bacteria, get 1mL bacterium enrichment liquid in the 2mL centrifuge tube of sterilizing, the centrifugal 5min of 10000rpm outwells supernatant liquor, and in the 1mL bacterial lysate, vortex concussion 15s mixes 70 ℃ of water-bath 10min with resolution of precipitate;
Described bacterial lysate consists of: 50mM Tris-HCl, 10mM EDTA, 1% SDS, 5mg/mL Proteinase K, pH8.0;
Or for gram-positive microorganism, get 1mL bacterium enrichment liquid in the 2mL centrifuge tube of sterilizing, the centrifugal 5min of 10000rpm, outwell supernatant liquor, precipitation is dissolved in the 500 μ L buffer A, vortex concussion 15s mixes, behind 37 ℃ of water-bath 30min, add in the 500 μ L buffer B, vortex concussion 15s mixes 70 ℃ of water-bath 10min;
Described buffer A consists of: contain 50mM Tris-HCl, 10mM EDTA, 1.2% Triton, 20mg/mL N,O-Diacetylmuramidase, pH8.0;
Described buffer B consists of: contain 1% SDS, 5mg/mL Proteinase K, pH8.0;
(3) preparation of adsorption-buffering liquid and interpolation:
The PEG/NaCl aqueous solution of preparation adsorption-buffering liquid 10%/6M, wherein polyethylene glycol 6000 mass concentration 10%, NaCl concentration 6M;
In step (2) gained 1mL bacterial lysate, add the magnetic nanometer particle congery 10 μ L that prepare, add the adsorption-buffering liquid PEG/NaCl aqueous solution 500 μ L again, make the NaCl final concentration be controlled to be 2M, turn upside down for several times, mix, concussion reaction 10min, the magnetic nano-particle of acquisition absorption bacterial genomes DNA;
(4) magnet absorption: will be adsorbed with magnetic nano-particle and the solution separating of bacterial genomes DNA with magnet, and outwell supernatant liquor;
(5) with 70% ethanol rinsing: clean the magnetic nano-particle twice that is adsorbed with bacterial genomes DNA with 70% ethanol rinsing liquid, room temperature is placed 0.5h, makes the residual ethanol volatilization fully;
(6) use the TE buffer solution elution: add 50 μ LTE damping fluids, 10min is hatched in 65 ℃ of water-baths behind the mixing, and magnetic separates, and gets supernatant, is the bacterial genomes dna solution, is stored in-20 ℃, and is standby;
Described TE damping fluid is: the mixing solutions that contains 10mM Tris-HCl, 1mM EDTA, pH8.0.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399774A (en) * | 2011-09-22 | 2012-04-04 | 王利兵 | Method for preparing Escherichia coli O157:H7 genome deoxyribonucleic acid (DNA) serving polymerase chain reaction (PCR) standard substance |
| CN103627703A (en) * | 2013-12-19 | 2014-03-12 | 涂祖新 | Total DNA (deoxyribonucleic acid) extraction method and kit for synchronously removing humic acid and mycoprotein |
| CN103937907A (en) * | 2014-05-14 | 2014-07-23 | 中华人民共和国北京出入境检验检疫局 | Plasmodium falci parum nano magnetic separation real-time fluorescence quantitative PCR (polymerase chain reaction) detection kit and nucleotide sequence |
| CN112501161A (en) * | 2020-12-23 | 2021-03-16 | 华南师范大学 | Double-magnetic-particle-intervention DNA extraction and purification method |
-
2010
- 2010-12-23 CN CN201010602179XA patent/CN102121002A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399774A (en) * | 2011-09-22 | 2012-04-04 | 王利兵 | Method for preparing Escherichia coli O157:H7 genome deoxyribonucleic acid (DNA) serving polymerase chain reaction (PCR) standard substance |
| CN103627703A (en) * | 2013-12-19 | 2014-03-12 | 涂祖新 | Total DNA (deoxyribonucleic acid) extraction method and kit for synchronously removing humic acid and mycoprotein |
| CN103627703B (en) * | 2013-12-19 | 2017-10-17 | 江西省科学院微生物研究所 | The synchronous Total DNA extraction method and kit for removing humic acid and mycoprotein |
| CN103937907A (en) * | 2014-05-14 | 2014-07-23 | 中华人民共和国北京出入境检验检疫局 | Plasmodium falci parum nano magnetic separation real-time fluorescence quantitative PCR (polymerase chain reaction) detection kit and nucleotide sequence |
| CN112501161A (en) * | 2020-12-23 | 2021-03-16 | 华南师范大学 | Double-magnetic-particle-intervention DNA extraction and purification method |
| CN112501161B (en) * | 2020-12-23 | 2023-03-28 | 华南师范大学 | Double-magnetic-particle-intervention DNA extraction and purification method |
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Application publication date: 20110713 |