CN106244584A - A kind of method of rapid extraction bacterial genomes DNA - Google Patents
A kind of method of rapid extraction bacterial genomes DNA Download PDFInfo
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- CN106244584A CN106244584A CN201610854824.4A CN201610854824A CN106244584A CN 106244584 A CN106244584 A CN 106244584A CN 201610854824 A CN201610854824 A CN 201610854824A CN 106244584 A CN106244584 A CN 106244584A
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- water
- plasma
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- dna
- bacterial genomes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Abstract
A kind of method that the invention discloses rapid extraction bacterial genomes DNA, utilizes low-temperature plasma activation water to react with antibacterial bacterium solution, and direct cell lysis wall discharges bacterial genomes DNA.The inventive method is simple, low cost, and the time is short, can bacterial genomes DNA in on-the-spot rapid extraction sample, for molecular Biological Detection.
Description
Technical field
The invention belongs to microbiological specimens pre-treatment field, be specifically related to a kind of for on-the-spot rapid extraction sample bacterium base
Because of the method organizing DNA, produce plasma mainly by electrion and directly mix with sterilized water, generate plasma-activated
Water, by with include that air, water body and human sample etc. mix, rapid extraction sample DNA, tie with current gene tester
Bacteria Detection can be rapidly completed after conjunction.
Background technology
Along with the progress of Biochemistry and Molecular Biology technology, nucleic acid is increasingly extensive in the application of microorganism field, core
Acid detection technique quickly grows.Such as, ring mediated isothermal amplification (loop-mediated isothermal
Amplification, LMAP) etc. Fast Detection Technique occur, the pattern detection time is greatly shortened, and detection limit reduces.This is to sample
This pre-treatment proposes new requirement, and low cost, method for extracting nucleic acid the shortest, easy and simple to handle are increasingly becoming research heat
Point.
At present, bacterial genomes DNA extraction method has special DNA extraction RNA isolation kit, alkaline lysis, boiling method, SDS to split
Solution and other wall-breaking methods (ultrasonication, broken, the multigelation of grinding etc.).Alkaline lysis have simple and direct, that purity is high etc. is excellent
Point, is one of the general extraction methods of current most widely used bacterial genomes DNA, but the method extraction time is longer, logical
Often need more than two hours, and there is certain corrosivity；Application specific DNA extraction kit extracts bacterial genomes DNA, effect
The best, but relatively costly, and clinical practice is restricted；The boiling method time is short, simple to operate, and yield is relatively low, the defect of its maximum
It is to the poor effect extracting gram positive bacteria genomic DNA；SDS cracking process and other cleavage methods need complicated cracking
System, not only prepares trouble, and Some Drugs is poisonous, and operational danger is big, Some Drugs and relevant enzyme reagent price in addition
Costliness, is therefore subject to certain restrictions in application.
The present invention uses low-temperature plasma activation water (Plasma prepared by atmosphere pressure plasma jet flow spray gun
Activated Water, PAW), this kind of treated water has strong oxidizing property, with antibacterial bacterium solution hybrid reaction after, can lead to
Cross physical method and realize the cracking to cell, extract bacterial genomes DNA, there is the feature such as simplicity, quick, material easily acquisition,
It is substantially shorter the time that sample of nucleic acid extracts, significantly reduces the expense of DNA extraction and the dependence to particular device.State at present
Inside and outside the most do not take this method extract bacterial genomes DNA.
Summary of the invention
A kind of method that it is an object of the invention to provide rapid extraction bacterial genomes DNA, it is thin that the method can be used for cracking
Cell wall, discharges nucleic acid from cell, and gained nucleic acid samples can be used for molecular Biological Detection, and such as ring mediated isothermal expands
Increase methods such as (Loop-mediated Isothermal Amplification, LAMP).
The technical solution used in the present invention is as follows:
A kind of method of rapid extraction bacterial genomes DNA, comprises the following steps:
1) low-temperature plasma activation water (PAW) is prepared；
2) by step 1) activated water prepared mixes with bacterium solution, concussion reaction certain time, can realize splitting of cell wall
Solve and the extraction of DNA.
Above-mentioned steps 1) preferably employ atmospheric pressure plasma (atmospheric pressure cold plasma) and penetrate
Low-temperature plasma activation water prepared by stream spray gun.Wherein, using high-frequency and high-voltage alternating current as atmos low-temperature plasma jet
The excitation power source of spray gun, optimization power supply input voltage is 50～100V, and electric current adjusts in real time, typically 0.2～0.5A.Carrier gas is excellent
Choosing uses 5%V/V oxygen and 95%V/V argon or the mixed gas of nitrogen, and the low temperature plasma of generation sprays from spray tip
Go out.Nozzle is placed in sterilized water ullage (distance liquid level about 2cm), excites low temperature plasma to react with water, when water
Between pH to 3～5, oxidation-reduction potential (OPR) value, between 400～500mV, is preparation and completes, when generally controlling reaction
Between 10～30min, generate low-temperature plasma activation water (PAW), for subsequent experimental after concussion mixing.
Above-mentioned steps 2) in, preferably PAW and bacterium solution by volume mix at 1: 1, concussion reaction 5～20min.
Use the water crossed of Low Temperature Plasma Treating to have a strong oxidizing property, and have that pH value is low, oxidation-reduction potential (ORP)
Value height, and NO can be stored for a long time2 -、NO3 -With H2O2Etc. feature.The present invention utilizes low-temperature plasma activation water and antibacterial bacterium solution
Reaction, direct cell lysis wall, discharge bacterial genomes DNA.The inventive method is simple, low cost, and the time is short, can show
Bacterial genomes DNA in the rapid extraction sample of field, for molecular Biological Detection.
Accompanying drawing explanation
LAMP testing result comparison diagram after Fig. 1: embodiment 1 plasma-activated water extraction P.aeruginosa DNA.
LAMP testing result comparison diagram after Fig. 2: embodiment 2 plasma-activated water extraction pneumococcal dna.
Detailed description of the invention
The present invention is expanded on further below in conjunction with embodiment, it will be understood by those skilled in the art that following example are only used for
The present invention is described rather than limits the scope of the invention.
Embodiment 1: plasma-activated water is used for extracting gram negative bacteria-P.aeruginosa DNA
(1) plasma-activated water is prepared: the excitation power source input voltage of atmos low-temperature plasma jet spray gun
50V, electric current 0.20A, carrier gas uses argon (containing 5%V/V oxygen), flow velocity 5L/min, nozzle is placed in 50mL sterilized water liquid level
Upper 2cm, excites low temperature plasma to react with water, and the response time is 30min, generates low-temperature plasma activation water (PAW), shake
Swing mixing 10s.
(2) Pseudomonas aeruginosa bacterium solution is prepared: use LB culture medium to cultivate 24 hours at 37 DEG C.After bacterium colony grows, use
It is scraped off by 20mL sterilized water from media surface, is placed in 50mL centrifuge tube, and under 7000rpm, centrifugal 7min, abandons supernatant
Rear addition 20mL sterilized water, concussion mixing；Repeat the above steps twice, is uniformly mixed so as to obtain pending after being eventually adding 20mL sterilized water
Bacterium solution.
(3) gradient processes: is diluted by original bacteria liquid, obtains 100, 10-1, 10-2, 10-3, 10-4, 10-5The bacterium solution of concentration.
(4) bacterium solution of above-mentioned 6 Concentraton gradient the most by volume with PAW mix at 1: 1, shake and react 20min.
(5) use sky root bacterial genomes to extract test kit (Tian Gen bio tech ltd, Beijing) simultaneously extract step
Suddenly (3) gained gradient bacterium solution, as comparison.
(6) using the method for ring mediated isothermal amplification (LAMP) to expand above-mentioned reactant liquor, reaction system is 25 μ L,
Comprise 2 μ L DNA profilings, FIP and BIP of 1.6 μMs, F3 and B3 of 0.2 μM, LF and LB of 0.8 μM, 8U Bst enzyme, and 12.5
2 × the RM of μ L, uses ddH2O polishing volume.By mixed system as in transmissometer (LA-500, Kyoto, Japan), 64 DEG C anti-
Answer 60min, last 80 DEG C of heat treated 2min.
Result is as it is shown in figure 1, during high concentration, use PAW to process G-The DNA sample that antibacterial (Pseudomonas aeruginosa) obtains,
The detection time of LAMP reaction, less than traditional adsorption column method, shows that the DNA concentration that the method is extracted is higher, and bacterial concentration is slightly lower
Time, the efficiency using PAW method to process antibacterial is slightly below kit method.
Embodiment 2: plasma-activated water is used for extracting gram positive bacteria-pneumococcal dna
(1) plasma-activated water is prepared: the excitation power source input voltage of atmos low-temperature plasma jet spray gun
50V, electric current 0.20A, carrier gas uses argon (containing 5%V/V oxygen), flow velocity 5L/min, nozzle is placed in 50mL sterilized water liquid level
Upper 2cm, excites low temperature plasma to react with water, and the response time is 30min, generates low-temperature plasma activation water (PAW), shake
Swing mixing 10s.
(2) streptococcus pneumoniae bacterium solution is prepared: use LB culture medium to cultivate 24 hours at 37 DEG C.After bacterium colony grows, use
It is scraped off by 20mL sterilized water from media surface, is placed in 50mL centrifuge tube, and under 7000rpm, centrifugal 7min, abandons supernatant
Rear addition 20mL sterilized water, concussion mixing；Repeat the above steps twice, is uniformly mixed so as to obtain pending after being eventually adding 20mL sterilized water
Bacterium solution.
(3) gradient dilution processes: by original bacteria liquid gradient dilution, respectively obtain 1,10-1, 10-2, 10-3, 10-4, 10-5Concentration
Bacterium solution.
(4) bacterium solution of above-mentioned 6 Concentraton gradient the most by volume with PAW mix at 1: 1, shake and react 20min.
(5) use sky root bacterial genomes to extract test kit (Tian Gen bio tech ltd, Beijing) simultaneously extract step
Suddenly (3) gained gradient bacterium solution, as comparison.
(6) using the method for ring mediated isothermal amplification (LAMP) to expand above-mentioned reactant liquor, reaction system is 25 μ L,
Comprise 2 μ L DNA profilings, FIP and BIP of 1.6 μMs, F3 and B3 of 0.2 μM, LF and LB of 0.8 μM, 8U Bst enzyme, and 12.5
2 × the RM of μ L, uses ddH2O polishing volume.By mixed system as in transmissometer (LA-500, Kyoto, Japan), 64 DEG C anti-
Answer 60min, last 80 DEG C of heat treated 2min.
Result is as in figure 2 it is shown, the method for bacterial genomes DNA that provides of the present invention, with the adsorption column method extraction side of standard
Method is compared, and material is simple and easy to get, low cost, and the response time is short, and on LAMP detection not impact, can be widely applied to carry
Take bacterial genomes DNA.
Claims (6)
1. a method for rapid extraction bacterial genomes DNA, comprises the following steps:
1) low-temperature plasma activation water is prepared；
2) by step 1) activated water prepared mixes with bacterium solution, concussion reaction certain time, it is achieved the cracking of cell wall and DNA's
Extract.
2. the method for claim 1, it is characterised in that step 1) use atmosphere pressure plasma jet flow spray gun to prepare low
Isothermal plasma activated water.
3. method as claimed in claim 2, it is characterised in that step 1) using high-frequency and high-voltage alternating current as atmos plasma
The excitation power source of body fluid jet spray gun, input voltage is 50～100V, and electric current is 0.2～0.5A.
4. method as claimed in claim 2, it is characterised in that described atmosphere pressure plasma jet flow spray gun is with 5%V/V oxygen
With the mixed gas of 95%V/V argon or nitrogen as carrier gas.
5. method as claimed in claim 2, it is characterised in that step 1) to utilize atmosphere pressure plasma jet flow spray gun to excite low
Isothermal plasma reacts with water, and between pH to 3～5 of water, oxidation-reduction potential value, between 400～500mV, obtains low temperature
Plasma-activated water.
6. method as claimed in claim 2, it is characterised in that step 2) in low-temperature plasma activation water with bacterium solution by volume
1: 1 mixing, concussion reaction 5～20min.
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| CN201610854824.4A CN106244584B (en) | 2016-09-27 | 2016-09-27 | A kind of method of rapidly extracting bacterial genomes DNA |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104150558A (en) * | 2014-08-05 | 2014-11-19 | 中山大学 | Method for preparing activated water through micro plasma |
| CN104707154A (en) * | 2015-02-13 | 2015-06-17 | 西安交通大学 | Dry-wet plasma sterilization device and method |
| CN104837349A (en) * | 2012-10-05 | 2015-08-12 | Ep科技有限公司 | Solutions and methods of making solutions to kill or deactivate spores, microorganisms, bacteria and fungus |
-
2016
- 2016-09-27 CN CN201610854824.4A patent/CN106244584B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104837349A (en) * | 2012-10-05 | 2015-08-12 | Ep科技有限公司 | Solutions and methods of making solutions to kill or deactivate spores, microorganisms, bacteria and fungus |
| CN104150558A (en) * | 2014-08-05 | 2014-11-19 | 中山大学 | Method for preparing activated water through micro plasma |
| CN104707154A (en) * | 2015-02-13 | 2015-06-17 | 西安交通大学 | Dry-wet plasma sterilization device and method |
Non-Patent Citations (3)
| Title |
|---|
| 倪盈 等: "低温等离子体杀菌的实验研究", 《环境工程学报》 * |
| 张灿 等: "水中细菌的低温等离子体灭活效果", 《环境与健康杂志》 * |
| 郭俭: "低温等离子体杀菌机理与活性水杀菌作用研究", 《中国博士学位论文全文数据库工程科技Ⅰ辑》 * |
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