CN106244584B - A kind of method of rapidly extracting bacterial genomes DNA - Google Patents
A kind of method of rapidly extracting bacterial genomes DNA Download PDFInfo
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- CN106244584B CN106244584B CN201610854824.4A CN201610854824A CN106244584B CN 106244584 B CN106244584 B CN 106244584B CN 201610854824 A CN201610854824 A CN 201610854824A CN 106244584 B CN106244584 B CN 106244584B
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- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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Abstract
The invention discloses the methods of rapidly extracting bacterial genomes DNA a kind of, are reacted using low-temperature plasma activation water with bacterial solution, Direct Pyrolysis cell wall releases bacterial genomes DNA.The method of the present invention is simple and easy, at low cost, and the time is short, can bacterial genomes DNA in live rapidly extracting sample, be used for molecular Biological Detection.
Description
Technical field
The invention belongs to microbiological specimens pre-treatment fields, and in particular to a kind of rapidly extracting sample bacterium base on site
Because of the method for group DNA, plasma mainly is generated using electrion and is directly mixed with sterile water, is generated plasma-activated
Water, by being mixed with including air, water body and human sample etc., rapidly extracting sample DNA, with current gene tester knot
Bacteria Detection can be rapidly completed after conjunction.
Background technique
With the progress of Biochemistry and Molecular Biology technology, nucleic acid is in microorganism field using increasingly extensive, core
Sour detection technique is quickly grown.For example, ring mediated isothermal amplification (loop-mediated isothermal
Amplification, LMAP) etc. Fast Detection Techniques occur, the pattern detection time greatly shortens, and detection limit reduces.This is to sample
This pre-treatment is put forward new requirements, and low cost, time-consuming method for extracting nucleic acid short, easy to operate are increasingly becoming research heat
Point.
Currently, bacterial genomes DNA extraction method has special DNA extracts kit method, alkaline lysis, boiling method, SDS to split
Solution and other wall-breaking methods (ultrasonication, the broken, multigelation of grinding etc.).Alkaline lysis has simple and direct, with high purity etc. excellent
Point is one of the general extraction methods of current most widely used bacterial genomes DNA, but this method extraction time is longer, leads to
Often need two hours or more, and there is certain corrosivity；Application specific DNA extraction kit extracts bacterial genomes DNA, effect
Fruit is good, but higher cost, and clinical application is restricted；The boiling method time is short, easy to operate, and yield is lower, maximum defect
It is to the ineffective of extraction gram-positive bacteria genomic DNA；SDS cracking process and other cleavage methods need complicated cracking
System not only prepares trouble, but also Some Drugs are toxic, and operational danger is big, furthermore Some Drugs and relevant enzyme reagent price
Valuableness, therefore be above subject to certain restrictions in application.
The present invention uses the low-temperature plasma activation water (Plasma of atmosphere pressure plasma jet flow spray gun preparation
Activated Water, PAW), there is this kind of treated water strong oxidizing property can lead to after bacterial solution hybrid reaction
Cracking of the physical method realization to cell is crossed, bacterial genomes DNA is extracted, has the characteristics that easy, quick, material easily obtains,
It is substantially shorter the time of sample of nucleic acid extraction, significantly reduces the expense that DNA is extracted and the dependence to particular device.State at present
It is inside and outside that this method is taken to extract bacterial genomes DNA not yet.
Summary of the invention
The object of the present invention is to provide the method for rapidly extracting bacterial genomes DNA a kind of, this method can be used for cracking carefully
Cell wall releases nucleic acid from cell, and gained nucleic acid samples can be used for molecular Biological Detection, such as ring mediated isothermal expands
Increase the methods of (Loop-mediated Isothermal Amplification, LAMP).
The technical solution adopted by the invention is as follows:
A kind of method of rapidly extracting bacterial genomes DNA, comprising the following steps:
1) low-temperature plasma activation water (PAW) is prepared；
2) activated water prepared by step 1) is mixed with bacterium solution, splitting for cell wall can be realized in concussion reaction certain time
The extraction of solution and DNA.
Above-mentioned steps 1) preferably penetrated using atmospheric pressure plasma (atmospheric pressure cold plasma)
Stream spray gun prepares low-temperature plasma activation water.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 are 50~100V, and electric current adjusts in real time, generally in 0.2~0.5A.Carrier gas is excellent
Choosing uses the mixed gas of 5%V/V oxygen and 95%V/V argon gas or nitrogen, and the low temperature plasma of generation is sprayed from spray tip
Out.Nozzle is placed in sterile water ullage (apart from liquid level 2cm or so), excitation low temperature plasma is reacted with water, when water
Between pH to 3~5, oxidation-reduction potential (OPR) value between 400~500mV, as prepares completion, when usually control is reacted
Between in 10~30min, generate low-temperature plasma activation water (PAW), concussion is used for subsequent experimental after mixing.
Above-mentioned steps 2) in, preferably PAW is mixed with bacterium solution by volume 1: 1,5~20min of concussion reaction.
There is strong oxidizing property, and the low, oxidation-reduction potential (ORP) with pH value using the water that Low Temperature Plasma Treating is crossed
Value is high, and can store NO for a long time2 -、NO3 -With H2O2The features such as.The present invention utilizes low-temperature plasma activation water and bacterial solution
Reaction, Direct Pyrolysis cell wall release bacterial genomes DNA.The method of the present invention is simple and easy, at low cost, and the time is short, can show
Bacterial genomes DNA in the rapidly extracting sample of field, is used for molecular Biological Detection.
Detailed description of the invention
Fig. 1: LAMP testing result compares figure after the plasma-activated water of embodiment 1 extracts P.aeruginosa DNA.
Fig. 2: LAMP testing result compares figure after the plasma-activated water of embodiment 2 extracts pneumococcal dna.
Specific embodiment
Below with reference to embodiment, the present invention is further explained, it will be understood by those skilled in the art that following embodiment is only used for
Illustrate the present invention rather than limits the scope of the invention.
Embodiment 1: plasma-activated water is for extracting Gram-negative bacteria-P.aeruginosa DNA
(1) plasma-activated water: the excitation power source input voltage of atmos low-temperature plasma jet spray gun is prepared
50V, electric current 0.20A, carrier gas use argon gas (oxygen containing 5%V/V), and nozzle is placed in the sterile water level of 50mL by flow velocity 5L/min
Upper 2cm, excitation low temperature plasma are reacted with water, and reaction time 30min is generated low-temperature plasma activation water (PAW), shake
It swings and mixes 10s.
(2) it prepares pseudomonas aeruginosa bacterium solution: being cultivated 24 hours at 37 DEG C using LB culture medium.After bacterium colony is grown, use
20mL sterile water scrapes off it from media surface, is placed in 50mL centrifuge tube, and 7min is centrifuged under 7000rpm, abandons supernatant
20mL sterile water is added afterwards, concussion mixes；Repeat the above steps twice, be eventually adding after 20mL sterile water be uniformly mixed so as to obtain it is to be processed
Bacterium solution.
(3) gradient is handled: original bacteria liquid being diluted, obtains 100, 10-1, 10-2, 10-3, 10-4, 10-5The bacterium solution of concentration.
(4) bacterium solution of above-mentioned 6 concentration gradients is pressed volume 1: 1 respectively to mix with PAW, concussion reaction 20min.
(5) step is extracted using Tiangeng bacterial genomes extracts kit (Tiangeng Biotechnology Co., Ltd, Beijing) simultaneously
Suddenly gradient bacterium solution obtained by (3), as control.
(6) above-mentioned reaction solution being expanded using the method for ring mediated isothermal amplification (LAMP), reaction system is 25 μ L,
Include 2 μ L DNA profilings, 1.6 μM of FIP and BIP, 0.2 μM of F3 and B3,0.8 μM of LF and LB, 8U Bst enzyme and 12.5
2 × 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 treatment 2min.
As a result as shown in Figure 1, when high concentration, PAW is used to handle G-The DNA sample that bacterium (pseudomonas aeruginosa) obtains,
The detection time of LAMP reaction is less than traditional adsorption column method, and the DNA concentration for showing that this method is extracted is higher, and bacterial concentration is slightly lower
When, the efficiency using PAW method processing bacterium is slightly below kit method.
Embodiment 2: plasma-activated water is for extracting gram-positive bacteria-pneumococcal dna
(1) plasma-activated water: the excitation power source input voltage of atmos low-temperature plasma jet spray gun is prepared
50V, electric current 0.20A, carrier gas use argon gas (oxygen containing 5%V/V), and nozzle is placed in the sterile water level of 50mL by flow velocity 5L/min
Upper 2cm, excitation low temperature plasma are reacted with water, and reaction time 30min is generated low-temperature plasma activation water (PAW), shake
It swings and mixes 10s.
(2) it prepares streptococcus pneumonia bacterium solution: being cultivated 24 hours at 37 DEG C using LB culture medium.After bacterium colony is grown, use
20mL sterile water scrapes off it from media surface, is placed in 50mL centrifuge tube, and 7min is centrifuged under 7000rpm, abandons supernatant
20mL sterile water is added afterwards, concussion mixes；Repeat the above steps twice, be eventually adding after 20mL sterile water be uniformly mixed so as to obtain it is to be processed
Bacterium solution.
(3) gradient dilution is handled: by original bacteria liquid gradient dilution, respectively obtaining 1,10-1, 10-2, 10-3, 10-4, 10-5Concentration
Bacterium solution.
(4) bacterium solution of above-mentioned 6 concentration gradients is pressed volume 1: 1 respectively to mix with PAW, concussion reaction 20min.
(5) step is extracted using Tiangeng bacterial genomes extracts kit (Tiangeng Biotechnology Co., Ltd, Beijing) simultaneously
Suddenly gradient bacterium solution obtained by (3), as control.
(6) above-mentioned reaction solution being expanded using the method for ring mediated isothermal amplification (LAMP), reaction system is 25 μ L,
Include 2 μ L DNA profilings, 1.6 μM of FIP and BIP, 0.2 μM of F3 and B3,0.8 μM of LF and LB, 8U Bst enzyme and 12.5
2 × 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 treatment 2min.
As a result as shown in Fig. 2, the method for bacterial genomes DNA provided by the invention, the adsorption column method extraction side with standard
Method is compared, and material is simple and easy to get, at low cost, and the reaction time is short, and is not influenced on LAMP detection, can be widely applied to mention
Take bacterial genomes DNA.
Claims (3)
1. a kind of method of rapidly extracting bacterial genomes DNA, comprising the following steps:
1) low-temperature plasma activation water is prepared: anti-using atmosphere pressure plasma jet flow spray gun excitation low temperature plasma and water
Low-temperature plasma activation water should be generated, wherein the excitation power source of atmosphere pressure plasma jet flow spray gun is high-frequency alternating current, defeated
Entering voltage is 50 ~ 100V, and electric current is 0.2 ~ 0.5A, and carrier gas is the mixed gas of 5%V/V oxygen and 95%V/V argon gas or nitrogen；
2) activated water prepared by step 1) is mixed, concussion reaction certain time with bacterium solution, realizes the cracking of cell wall with DNA's
It extracts.
2. the method as described in claim 1, which is characterized in that step 1) is excited low using atmosphere pressure plasma jet flow spray gun
Isothermal plasma is reacted with water, and between the pH to 3 ~ 5 of water, oxidation reduction potential value obtains low temperature between 400 ~ 500 mV
Plasma-activated water.
3. the method as described in claim 1, which is characterized in that low-temperature plasma activation water and bacterium solution press volume in step 2
1: 1 mixing, 5 ~ 20min of concussion reaction.
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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 |
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- 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 |
|---|
| 低温等离子体杀菌机理与活性水杀菌作用研究;郭俭;《中国博士学位论文全文数据库工程科技Ⅰ辑》;20160515(第05期);第B024-2页 |
| 低温等离子体杀菌的实验研究;倪盈 等;《环境工程学报》;20091130;第3卷(第11期);第1951-1955页 |
| 水中细菌的低温等离子体灭活效果;张灿 等;《环境与健康杂志》;20131231;第30卷(第12期);第1081-1083页 |
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