CN102719425A - Method for extracting nucleic acid from activated carbon biological membranes in drinking water treatment - Google Patents
Method for extracting nucleic acid from activated carbon biological membranes in drinking water treatment Download PDFInfo
- Publication number
- CN102719425A CN102719425A CN2012101875478A CN201210187547A CN102719425A CN 102719425 A CN102719425 A CN 102719425A CN 2012101875478 A CN2012101875478 A CN 2012101875478A CN 201210187547 A CN201210187547 A CN 201210187547A CN 102719425 A CN102719425 A CN 102719425A
- Authority
- CN
- China
- Prior art keywords
- activated carbon
- nucleic acid
- biological activated
- drinking water
- ultrasonic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for extracting nucleic acid from activated carbon biological membranes in drinking water treatment and relates to the technical field of microbiology of biological activated carbon process units in environmental engineering drinking water deep treatment. The method includes: firstly, subjecting activated carbon particles to dehumification by the aid of dehumification buffer solution; and then performing water bath and ultrasonic elution. The method specifically includes: subjecting activated carbon to dehumification by the aid of the dehumification buffer solution obtained by mixing NaOH solution or NaOH with polyvinylpyrrolidone, and extracting the microbial nucleic acid on the biological activated carbon after ultrasonic elution and chemical cleavage. The total DNA (deoxyribonucleic acid) length is larger than 20 Kb so as to completely meet the requirements of PCR (polymerase chain reaction) analysis. According to the method, the problem of difficulty in elution resulted from low biomass of the biological activated carbon and close combination of the biological activated carbon with carrier particles is solved, and adverse effect of humic acid on microbial nucleic acid extraction is weakened, so that microbial cells are sufficient in cleavage. The method is simple and convenient to operate and high in DNA output and purity.
Description
Technical field
The present invention relates to the microbial nucleic acids process for extracting on the biological activated carbon in a kind of drinking water treatment, belong to the drinking water deep processing technology field.
Background technology
Gac is owing to have good adsorption performance and stable chemical performance; Can anti-strong acid and highly basic, can stand water logging and high temperature, lighter than water; Be the porous hydrophobic adsorbent, so be widely used in stink, colourity, the natural and artificial synthetic organic materials of removing in the tap water.The microbial film that is the main body with mikrobe and the outer polymer of born of the same parents thereof that adheres on the gac is the biodegradable major portion of objectionable impurities in the water body.Therefore the health risk that gac biological degradation mechanism and mikrobe leakage caused during identification and analysis activated carbon surface biological community structure was handled for clear and definite drinking water deep with distribution is significant.
Traditional microorganism detection is based upon on the mikrobe separation and Culture basis, and sense cycle is long, complicated operation, and specificity is not strong.Simultaneously a large amount of mikrobes in the environment can't be realized artificial culture, and perhaps structure of community so only depends on existing microorganism culturing technology can not reflect the microbial diversity that belongs to environment all sidedly because the change of living environment changes in culturing process.So Protocols in Molecular Biology becomes the main means of research biological activated carbon microflora.
(Cetyilrt methyl ammonium bromide, CTAB) under certain salinity, forming deposition extraction DNA with nucleic acid is the method for using always to utilize cetyl trimethylammonium bromide.CTAB is a kind of cationic detergent, can form mixture with nucleic acid.This mixture is at high salt (> 0.7mol/L) solvable under the concentration, and stable existence, when salt concn was reduced to 0.1~0.15mol/L, CTAB-nucleic acid complexes solubleness reduced and forms deposition, and most protein and polysaccharide etc. still are dissolved in the solution.Through the organic solvent extracting, add ethanol sedimentation behind the impurity such as removal protein, polysaccharide, phenols separate nucleic acid is come out then, wash away CTAB at last and get final product.Sodium lauryl sulphate (sodium dodecyl sulfate; SDS) be a kind of AS; Can interact with phosphatide in the microbial film and lipoprotein and destroy membrane structure, space structure that simultaneously also can destructive enzyme influences the catalytic activity of enzyme.When extracting DNA, the SDS (more than 0.5%) that often utilizes high density under comparatively high temps (55~65 ℃) lysing cell and passivation nucleicacidase to reduce the degraded of DNA.
Yet, because the living weight on the biological activated carbon of processing drinking water is few relatively, and tight with the carrier granule combination, so thalline is difficult to directly elute from gac.On the other hand; The humic acid that adsorbs on the gac is difficult for taking place biological degradation under field conditions (factors); Show multiple physico-chemical properties such as polarity, oxidation-reduction quality, complexing and absorption again; In the DNA extraction process of classics, mikrobe is swept along wherein, stop the infiltration of denaturing agents such as SDS and suppress the activity of N,O-Diacetylmuramidase and Proteinase K etc., influence the cracking of lysis buffer mikrobe with interaction such as clay and metals ion.The xanthohumic acid and the ulmic acid of stripping can be oxidized to quinone after alkaline bleach liquor cleavage liquid is handled simultaneously, can be reduced to phenol again, can cause part DNA to lose with the phenol entering organic phase of oxidation.Even adopt different methods such as dialysis, IX, density gradient centrifugation, chromatography and electrophoresis that the nucleic acid crude extract is further purified; Still have the close humic acid component of some physico-chemical properties and nucleic acid to coexist with it, thereby the subsequent P CR amplification that quantitatively reaches that has influence on nucleic acid wait technical process.Traditional DNA extraction method and commercially available DNA extraction test kit all can not effectively address the above problem, and therefore how before lysing cell, to adopt pre-treatment and the effective microbe elution process of removing humic acids just to become the key that obtains high yield and highly purified DNA from biological activated carbon.
Summary of the invention
The object of the invention be to solve in the prior art since on the biological activated carbon living weight few and combine the wash-out difficult problem that closely causes with carrier granule; Reduce humic acid microbial nucleic acids is extracted disadvantageous effect; Through taking off rotten the processing and the UW elution process; The method of microbial nucleic acids on a kind of effective extraction biological activated carbon is provided, all reaches the dna fragmentation that is applicable to the downstream molecules biologic operation to obtain output and purity.
Technical scheme of the present invention is following:
The biomembranous method for extracting nucleic acid of gac in a kind of drinking water treatment is characterized in that this method comprises the steps:
1) biological activated carbon take off rotten the processing: is 1: 8~1: 15 to mix with taking off rotten damping fluid by the mass/volume ratio with biological activated carbon; Take off rotten damping fluid and adopt NaOH solution; Or the mixing solutions of employing NaOH and Vinylpyrrolidone polymer; Mix back vortex vibration, centrifugal treating again obtains taking off the biological activated carbon after the corruption;
2) the ultrasonic wash-out of biological activated carbon is handled: the gac that will take off after the corruption is dissolved in the deionized water, and vortex vibration and the ultrasonic wash-out of the laggard row of normal temperature water-bath are handled;
3) with step 2) in the centrifugal supernatant of abandoning of suspension liquid after ultrasonic carry out purifying and extract, promptly obtain DNA;
In the technique scheme, the volumetric molar concentration of taking off rotten damping fluid NaOH in the step 1) is 50mmol/L~200mmol/L, and Vinylpyrrolidone polymer and NaOH mass ratio are 5: 1 to 10: 1 in the said mixing solutions; Centrifugal speed is that centrifugation time is at least 5min more than the 1000rpm in the step 1); Step 2) ultrasonic elution time is 1min~5min in, and ultrasonic frequency is 45~100kHz.
Of the present invention having the following advantages and the high-lighting effect:
1. take off rotten damping fluid and take off rotten ability height, reduced the disadvantageous effect that humic acid extracts microbial nucleic acids.2. the supersound process gac has overcome on the drinking water biological active carbon few and combine the wash-out difficulty that closely causes with carrier granule because of living weight.
In a word, the present invention has overcome the defective of traditional method for extracting nucleic acid (DNA), and simple to operate, can obtain high yield and highly purified DNA from biological activated carbon.
Description of drawings
Fig. 1 extracts total DNA electrophorogram among 6 embodiment.Electrophoresis band 1 is λ-Hind III digest DNA Marker, and electrophoresis band 2 ~ 7 is respectively 1 ~ 6 embodiment and extracts the total DNA of acquisition.
Fig. 2 is the PCR electrophorogram that extracts total DNA among 6 embodiment.Electrophoretic band 1 is M100bp DNA Ladder, and electrophoretic band 2 ~ 7 is respectively 1~6 embodiment and extracts the pcr amplification that obtains total DNA.
Embodiment
For understanding the present invention better, further specify through specific embodiment below.
The biomembranous method for extracting nucleic acid concrete steps of gac are following in the drinking water treatment of the present invention:
1) biological activated carbon take off rotten the processing: with biological activated carbon with take off rotten damping fluid and mix by different mass/volume ratio, generally biological activated carbon is to mix in 1: 8~1: 15 with taking off rotten damping fluid by the mass/volume ratio; Take off rotten damping fluid and adopt NaOH solution, or adopt the mixing solutions of NaOH and PVP, mix the back vortex and vibrate, centrifugal treating again obtains taking off the biological activated carbon after the corruption; Centrifugal speed is that centrifugation time is at least 5min more than the 1000rpm; The volumetric molar concentration of taking off rotten damping fluid NaOH is 50mmol/L~200mmol/L, in the mixing solutions PVP and NaOH quality than scope for being 5: 1 to 10: 1.
2) the ultrasonic wash-out of biological activated carbon is handled: the biological activated carbon that will take off after the corruption is dissolved in the deionized water, and vortex vibration and the ultrasonic wash-out of the laggard row of normal temperature water-bath are handled; Ultrasonic frequency is generally 45~100kHz, and ultrasonic elution time is 1min~5min.
3) with step 2) in the centrifugal supernatant of abandoning of suspension liquid after ultrasonic, carry out nucleic acid purification and extract.Purifying extracts and adopts the commercial reagent box to extract, and also can adopt following classical way: be specially and get the ultrasonic back of 5ml suspension liquid, 1.3 * 10
4The centrifugal 5min of rpm discards supernatant, adds the 500 resuspended thalline of μ l TE damping fluid and changes in the centrifuge tube of a 1.5ml, adds the Proteinase K of 30 μ l 10%SDS and 15 μ l, and mixing is in 37 ℃ of incubation 1h.Add the abundant mixing of 100 μ l 5mol/L NaCl, add 80 μ l CTAB/NaCl solution again, behind the mixing in 65 ℃ of incubation 10min.Add equal-volume chloroform/primary isoamyl alcohol (24: 1), mixing gently vibrates.Centrifugal 4 ~ 5min transfers to supernatant in the new centrifuge tube, adds 0.6 volume Virahol, and room temperature leaves standstill 60min behind the mixing.1.3 * 10
4The centrifugal 20min of rpm, abandoning supernatant precipitates 70% washing with alcohol twice with 1ml, and the centrifugal ethanol of abandoning is dry a little in clean bench, heavily is dissolved in the 20 μ l TE damping fluids placing cryogenic refrigerator subsequent use, promptly obtains high-quality DNA.
Embodiment
The biological activated carbon sample is taken from waterworks, Beijing, and embodiment 1~3 adopts NaOH solution, and (50 ~ 200mmol/L) as taking off rotten damping fluid, and ultrasonic elution time is respectively 1min, 2min and 5min, extracts DNA; Embodiment 4~6 employing NaOH (50 ~ 200mmol/L)+PVP (PVP: NaOH 5: 2, w/w) mixing solutions is as taking off rotten damping fluid, and ultrasonic elution time is respectively 1min, 2min and 5min, extracts DNA.
The DNA extraction result shown in table one, the total DNA A that is obtained
260/ A
280Value is all more than 1.6, and explaining among total DNA does not have protein contamination, and some samples have part RNA to pollute in explanation more than 1.8, can add a little RNA enzyme and remove.Adopt NaOH solution (100mmol/L) as taking off rotten damping fluid, ultrasonic time increases to 5min by 1min and obtains the increase of DNA extraction amount.(PVP: NaOH 5: 2, w/w) mixing solutions is as taking off rotten damping fluid, institute's DNA extraction amount that obtains maximum when ultrasonic time is lacked (1min) to adopt NaOH (200mmol/L)+PVP.
WarpNaOH (50 ~ 200mmol/L)+PVP (PVP: NaOH 5: 2, w/w) mixing solutions
Take off The total DNA amount of sample extraction after rotten the processing be higher than withNaOH solution (50 ~ 200mmol/L) solution
Take off corruptionIt is big to use DNA extraction method provided by the invention to obtain output, the DNA that quality is high.
Table one: method and the effect of extracting total DNA compare
With obtaining DNA4ul, with 1.0% added the GoldViwe staining agent sepharose carry out electrophoresis, and the electrophoretic image of gained nucleic acid is analyzed with the Bio-Rad gel imaging system, as shown in Figure 1.6 kinds of modes all obtain the total DNA band of length more than 20Kb; And the two components of warp take off rotten damping fluid NaOH, and ((PVP: NaOH 5: 2, w/w) the DNA band of processing back sample is higher than the sample that only takes off rotten processing through NaOH solution (100mmol/L) to 50 ~ 200mmol/L)+PVP.
The pcr amplification product of obtaining DNA is detected electrophoretic image such as Fig. 2 through 1.2% agarose gel electrophoresis.Can find out that 6 embodiment all amplify target stripe, the total DNA that takes off the sample after rotten damping fluid is handled through NaOH+PVP is that template all can be compared with NaOH solution (100mmol/L) maximum for the PCR product that takes off rotten damping fluid and obtain.Explain and use DNA that method provided by the invention obtains to satisfy the downstream molecules biologic operation.
Claims (3)
1. the biomembranous method for extracting nucleic acid of gac in the drinking water treatment is characterized in that this method comprises the steps:
1) biological activated carbon take off rotten the processing: is 1: 8~1: 15 to mix with taking off rotten damping fluid by the mass/volume ratio with biological activated carbon; Take off rotten damping fluid and adopt NaOH solution; Or the mixing solutions of employing NaOH and Vinylpyrrolidone polymer; Mix back vortex vibration, centrifugal treating again obtains taking off the biological activated carbon after the corruption;
2) the ultrasonic wash-out of biological activated carbon is handled: the biological activated carbon that will take off after the corruption is dissolved in the deionized water, and vortex vibration and the ultrasonic wash-out of the laggard row of normal temperature water-bath are handled;
3) with step 2) in the centrifugal supernatant of abandoning of suspension liquid after ultrasonic, carry out nucleic acid purification and extract.
2. the biomembranous method for extracting nucleic acid of gac in a kind of drinking water treatment according to claim 1; It is characterized in that: the volumetric molar concentration of taking off rotten damping fluid NaOH in the step 1) is 50mmol/L~200mmol/L, and Vinylpyrrolidone polymer and NaOH mass ratio are 5: 1 to 10: 1 in the said mixing solutions.
3. the biomembranous method for extracting nucleic acid of gac in a kind of drinking water treatment according to claim 1 is characterized in that: centrifugal speed is that centrifugation time is at least 5min more than the 1000rpm in the step 1); Step 2) ultrasonic elution time is 1min~5min in, and ultrasonic frequency is 45 ~ 100kHz.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2012101875478A CN102719425A (en) | 2012-06-07 | 2012-06-07 | Method for extracting nucleic acid from activated carbon biological membranes in drinking water treatment |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2012101875478A CN102719425A (en) | 2012-06-07 | 2012-06-07 | Method for extracting nucleic acid from activated carbon biological membranes in drinking water treatment |
Publications (1)
Publication Number | Publication Date |
---|---|
CN102719425A true CN102719425A (en) | 2012-10-10 |
Family
ID=46945295
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN2012101875478A Pending CN102719425A (en) | 2012-06-07 | 2012-06-07 | Method for extracting nucleic acid from activated carbon biological membranes in drinking water treatment |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN102719425A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114085830A (en) * | 2022-01-12 | 2022-02-25 | 济凡生物科技(北京)有限公司 | Method for extracting microbial genome DNA (deoxyribonucleic acid) in complex sample |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102094003A (en) * | 2009-12-14 | 2011-06-15 | 中国科学院城市环境研究所 | Method for extracting DNA (deoxyribonucleic acid) of activated carbon biomembrane |
CN102286463A (en) * | 2011-06-29 | 2011-12-21 | 内蒙古大学 | High-efficiency humus-removing environment sample total DNA extraction method |
-
2012
- 2012-06-07 CN CN2012101875478A patent/CN102719425A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102094003A (en) * | 2009-12-14 | 2011-06-15 | 中国科学院城市环境研究所 | Method for extracting DNA (deoxyribonucleic acid) of activated carbon biomembrane |
CN102286463A (en) * | 2011-06-29 | 2011-12-21 | 内蒙古大学 | High-efficiency humus-removing environment sample total DNA extraction method |
Non-Patent Citations (3)
Title |
---|
侯彬等: "饮用水中腐植酸对人体健康的影响及去除方法", 《腐植酸》 * |
刘小琳等: "生物陶粒与生物活性炭上微生物群落结构的PCR-SSCP技术解析", 《环境科学》 * |
蒋云霞等: "不同提取缓冲液对红树林土壤DNA提取品质的影响", 《热带海洋学报》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114085830A (en) * | 2022-01-12 | 2022-02-25 | 济凡生物科技(北京)有限公司 | Method for extracting microbial genome DNA (deoxyribonucleic acid) in complex sample |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Lalov et al. | Treatment of waste water from distilleries with chitosan | |
CN102175606B (en) | Method for detecting acute biological toxicity of sewage | |
CN101717815B (en) | Legionnella rapid detecting and parting method | |
CN105524917A (en) | Kit for extracting blood genome DNA based on magnetic bead method and use method for kit | |
CN105802957B (en) | A method of extracting microorganism total DNA from the water sample of coal seam | |
CN103725670A (en) | Method for extracting nucleic acid from biological sample | |
JP2013519396A (en) | Nucleic acid extraction from complex matrices | |
CN110904098A (en) | Lysis binding solution and nucleic acid extraction by feces magnetic bead method | |
CN104014313A (en) | Improved wheat husk adsorbent | |
CN102822339A (en) | Method for precipitating anionic surfactant ions in presence of nucleic acids | |
Barnes et al. | Purification of catechol siderophores by boronate affinity chromatography: identification of chrysobactin from Erwinia carotovora subsp. carotovora | |
CN102206630A (en) | Method and kit for extracting total DNA of soil and sediment | |
CN101514339B (en) | Soil DNA extracting method directly used for analyzing microbial community structures | |
CN104911178A (en) | Method for simultaneously extracting microbial intracellular and extracellular DNAs (deoxyribonucleic acids) in sewage biological treatment water sample | |
CN104152436B (en) | DNA isolation and purification methods and its kit | |
CN103789300B (en) | A kind of extracting method of epoxy propane saponified wastewater active sludge macro genome DNA | |
CN105911191A (en) | Simultaneous extraction and purification method of four types of mycotoxin in rice | |
Yan et al. | Ultrasensitive quantification of pathogens in milliliters of beverage by filtration-based digital LAMP | |
CN104560953A (en) | Kit for rapidly extracting sludge microbial genome DNA and extracting method | |
CN111534509B (en) | Composition, reagent, kit and application for deep-sea microorganism in-situ cell lysis | |
CN106754895B (en) | Extract the method and kit of crude oil total dna | |
CN102807979A (en) | Method for extracting and pretreating microorganism deoxyribonucleic acid (DNA) on biological active carbon | |
CN102719425A (en) | Method for extracting nucleic acid from activated carbon biological membranes in drinking water treatment | |
CN104697831A (en) | Extraction method of biological membrane proteins and preparation method of electrophoresis sample | |
Liu et al. | Removal of polymerase chain reaction inhibitors by electromembrane extraction |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20121010 |