CN103740826A - Method for detecting genotoxic potential of heavy metal pollutants in soil - Google Patents
Method for detecting genotoxic potential of heavy metal pollutants in soil Download PDFInfo
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Abstract
The invention discloses a method for detecting genotoxic potential of heavy metal pollutants in soil. The method comprises the steps of 1, soil digestion; 2. preparation of microalgae cells; 3, glue spreading; 4. pyrolysis; 5. electrophoresis; 6. neutralization; 7. dyeing; and 8. DNA damage degree analysis and performing of ANOVA analysis by using statistic Origin 7.5 software. The comet image analysis is performed by adopting CASP software, the OTM (Olive Tail Moment) simultaneously reflects the DNA content and shape features in the comet in evaluation parameters and is a common index for quantifying the DNA damage degree. The method is an effective test method and can evaluate the genotoxic potential of heavy metals in the soil.
Description
Technical field
The invention belongs to Environmental Health genetic toxicology field, be specifically related to a kind of potential genotoxic method of detection heavy metal in soil.
Background technology
Soil is the important natural resources of human survival and development and the basis that whole TERRESTRIAL ECOSYSTEMS is rely and existed, and is again one of important environmental element.The soil of " health " is extremely important for agricultural sustainable development and the mankind's existence.But along with China's rapid development of economy, the problem of soil pollution is day by day serious, serious soil pollution particularly heavy metal contamination has become and has comprised at present urban soil in a denominator of interior many soil, and there have been 100,000 km in China according to estimates
2soil is contaminated, and in China's soil pollution heavy metal, As, Cd, Cr, Hg and the contour toxic heavy metal of Pb are seriously polluted.There are some researches show that heavy metal has stronger mutagenicity to biology.Therefore, harmful heavy metal pollutent in soil is investigated and extremely urgent to its genotoxic research, even someone proposes whether to contain Genotoxic in soil, should be the leading indicator of evaluating soil.So setting up a kind of genetoxic of effective testing method evaluation heavy metal in soil is an important process of environmental monitoring.
The biological detection of various environmental factor genetoxic effects has been occupied to very consequence in toxicologic study, and they have brought into play positive effect in environmental pollution monitoring and environment protection.A large amount of genetic analysis methods is used to identify sexual cell mutagenic compound, somatocyte mutagenic compound, potential carcinogen, and the various hereditary changes relevant to human health, and related method is over 200 kinds.The indicator terminal scope related according to genetoxic effect detection method, can be divided into three major types them.The first kind detects transgenation; Equations of The Second Kind detects chromosome aberration, comprises the abnormal change of chromosome structure and/or number; The sign that the 3rd class is measured DNA damage is as formation, sister chromatid exchange, somatocyte restructuring and the DNA bond rupture etc. of the exciting of DNA damage reparation, DNA adduct.The detection of transgenation mainly contains forward and reverse two classes.Forward mutation changes wild type gene, makes related gene inactivation and shows detectable phenotypic variation.On the contrary, reverse mutation is by sudden change, the gene function of inactivation in former muton to be recovered, thus performance wild-type phenotype.Chromosome aberration generally comprises the change of chromosome structure and number, and alteration of chromosome structure comprises rhexis, inversion, dystopy and repeats etc.Detect chromosome aberration and conventionally adopt Cytogenetic techniques.System for detection of chromosomal structural abnormality is generally in higher organism, especially mammalian body and vitro system.In recent years, due to the progress of molecular biological development and technology, make to have produced in genetic toxicology field many new genotoxicity testing method.Although detection in Gene Mutation amount " gold standard " is direct DNA sequencing, and automated DNA order-checking ability also has considerable progress, but order-checking remains that one more loaded down with trivial details, expense is high, needs the work of expensive device, be not suitable on a large scale DNA mutation in examination genome.
DNA is the genetic material in organism, and its stable normal Growth and Reproduction for organism is vital.Yet various physics and chemistry and biotic factor in environment---as ionizing rays, various oxygenant, bacterium etc. all may cause DNA damage.Single cell gel electrophoresis (singe cell gel electrophoresis assay, be called for short SCGE, have another name called Using Comet Assay, comet assay) being one is widely used in the unicellular DNA single chain of eukaryote, two strands, base point of instability and repairs the quantification detection means that postpones site, the method is easy, quick, sensitive, and every 109Da can detect the features such as 0.1 fracture and be widely applied.Compare with classical chromosome aberration, micronucleus, SCE, SCGE can also can make SCGE not only can study the biological effect under low dosage for the DNA analysis of dead cell for viable cell DNA detection, also can be for the biological effect under research high dosage; SCGE can provide the information of DNA repair ability again simultaneously, and this makes SCGE be suitable for very much evaluating the hereditary effect of tested material.
Single cell gel electrophoresis technique is pioneering in 1984 by Ostling, is improved respectively again thereafter by Singh and Olive, forms current alkalescence and neutral cracking two class methods.Although this technology principle, method are simple, its operating process is very difficult.In making the process of glue, generally on sanding slide glass, drip 2-3 layers of gels, with cover glass lid, press at every turn, when tearing off cover glass, very easily damage offset plate.In cracking thereafter, electrophoresis, washing process, gel soaks for a long time, rinses, though extremely light vibration, the levitating of also offset plate may being got excited, the damage of offset plate is often to the failure of an experiment.
SCGE technology is suitable for all eukaryotic cells theoretically, sample cell requirement is little, cell can be the cell of cultured cells system or cell tissue separation, and the cell of having used at present has root cells and blade cell and the yeast cell etc. of human lymphocyte, epidermic cell, hemocyte, inoblast, testicular cell, tumour cell, animal livers cell, plant.But the comet of relevant algae all rarely has report both at home and abroad.Algae is the important component part of ecotope, and algae, for SCGE, is expanded to the research object of SCGE.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of potential genotoxic method of detection heavy metal in soil, is just to provide specifically a kind of potential genotoxic method of utilizing micro-algae DNA damage to detect heavy metal in soil pollutent.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
The potential genotoxic method of detection heavy metal in soil pollutent, comprises the steps:
The pre-treatment of step 1. soil sample: gather pedotheque, air-dry after, the soil sieve that to cross diameter be 2mm, removes after plant residue, stone etc., grinds, all by 80 mesh sieves with agate mortar.Soil, after clearing up, with the warm dissolved residue of 1ml nitric acid, is then transferred to solution in 50ml volumetric flask, and constant volume, shakes up, and saves backup;
Step 3. paving glue:
With normal fusing point agarose (NMA) the PBS solution of 1%g/mL, (1%g/mL adds 1g NMA to step 3a. in 100mLPBS, identical to give a definition) 300--500 μ L bottoming on frosted slide glass, scrape off again, can make like this first layer glue more easily fix on slide glass;
Step 3b. prepares the first layer glue: normal fusing point agarose (NMA) the PBS solution of 60--120 μ L0.7%g/mL is dropped on the frosted slide glass that step 3a obtains to covered rapidly, 4 ℃ of curing 5-10min;
Step 3c. prepares second layer glue: to step 2, process low melting-point agarose (LMA) the PBS solution that adds 0.7%g/mL in the cell obtaining, and resuspended algae mud, piping and druming evenly becomes cell suspension, adjusts cell concn 1 * 10
5--1 * 10
6individual cell/mL.Then take out the solidifying slide of the first layer glue, gently pull out and remove lid, get 50--120 μ L and be added on the first layer glue, covered, 4 ℃ of curing 30-40min;
Step 3d. prepares the 3rd layer of glue: after the second layer solidifies, get the 3rd layer of glue of PBS solution paving of the normal fusing point agarose (NMA) of 50--120 μ L1%g/mL, after covered, put into refrigerator it is solidified;
Step 6. neutralization: with pH7.5,0.4mmol/L Tris-HCl damping fluid submergence slide glass, the each 5min of rinsing, in and 3 times;
Step 7. dyeing: drip ethidium bromide (EB) the 30 μ L of 50 μ g/mL on glue, add a cover cover glass, microscopy in 24h;
Step 8.DNA degree of injury is analyzed: with fluorescent microscope, at green glow, excite lower observation, take pictures, obtain after comet image, with CASP software analysis, measure the parameter of DNA migration: tail square (TM) and tail momentum (OTM), with it, evaluate the potential genetoxic of heavy metal in soil.
Step 9. experiment at least establish 3 parallel, get its mean value; Statistics is carried out ANOVA analysis with Origin7.5 software, and experimental group and control group are carried out to significant t-test, usings p<0.05 as significance foundation.
Comet image analysis adopts CASP software, and in evaluating, tail momentum (olive tail moment, OTM) has reflected DNA content and tail of a comet shape facility in comet simultaneously, is the common counter of quantification DNA damage degree.In addition, provide tail square (TM) parameter as with reference to index, to reflect DNA damage situation more comprehensively.Every slide glass is at least analyzed 50 cells.
Above-mentioned method, in step 1, described to clear up operating process as follows:
Take 0.2000g and cross the air-dry soil sample of 80 object in 30ml tetrafluoroethylene crucible, add 5ml HCl low-temperature heat, sample is tentatively decomposed, to be evaporated during to 2-3ml, take off slightly cold, then add 5ml nitric acid (technique is ultrapure), 3ml hydrofluoric acid, 3ml perchloric acid, after adding a cover, in the upper temperature heating of hot plate, within about 1 hour, (notice that temperature is too not high, in order to avoid solution is emerged), then uncap, continue heating silica removal, and often shake crucible to reach the good silicon effect that flies, when being heated to emit the white cigarette of dense perchloric acid, add a cover, black organic carbon compound is fully decomposed, after the black organism on crucible disappears, uncap to drive white cigarette and steam to content and be thick, depending on clearing up situation, can add again 2ml nitric acid, 2ml hydrofluoric acid, 2ml perchloric acid repeats said process, when white cigarette emits to the greatest extent and when content is thick again substantially, take off slightly cold, water rinses crucible cover and inwall.
Above-mentioned method, in step 2 and step 3, described microalgae cell is very thin Euglena cell.
Above-mentioned method, step 3a is preferably with normal fusing point agarose 350 μ L bottoming on frosted slide glass of 1%g/mL.
Above-mentioned method, step 3b preferably by the normal fusing point agarose drop of 100 μ L0.7%g/mL on the prefabricated slide glass obtaining of step 3a.
Above-mentioned method, in step 3c, preferably gets 75 μ L cell suspensions and is added drop-wise on the first layer glue.
Above-mentioned method in step 3d, preferably drips the normal fusing point agarose of 100 μ L1%g/mL on second layer glue.
Above-mentioned method, in step 4, preferably pyrolysis time is 20min.
Above-mentioned method, in step 5, preferably electrophoretic current is 200mA, and voltage is 20V, and electrophoresis time is 20min.
Beneficial effect: the present invention compared with prior art tool has the following advantages:
(1) Heavy Metal Pollution in Sediments in soil and water system, can affect the associated ecosystem, be accompanied by the physical chemistry bioprocesss such as absorption and sorption, leaching, heavy metal can migrate in vegetation and water body environment, thereby enter food chain, cause human and animal to be endangered, the serious ecosystem environment Economic development that even can affect periphery.Measure fast and accurately complex environment medium (soils and sediments) and need to relate to sample pre-treatments and instrument analytical method.Sample pre-treatments is that soil is cleared up, and makes dissolving metal in water, and instrumental analysis now conventional means is icp ms (ICP-MS).Yet instrumental analysis somewhat expensive and can only determine the content of heavy metal in pedotheque.The present invention for genetic toxicity test to algae, can judge in soil, whether there is Genotoxic by the solution of soil digestion fast.
(2) first clear up soil sample, the solution obtaining, for giving very thin Euglena contamination, is applied the comet of very thin Euglena, and the genetoxic of heavy metal in soil is studied, and sets up a kind of effective testing method, the genetoxic of grading heavy metal in soil.Soil is the important natural resources of human survival and development and the basis that whole TERRESTRIAL ECOSYSTEMS is rely and existed, and is again one of important environmental element.The soil of " health " is extremely important for agricultural sustainable development and the mankind's existence.But along with China's rapid development of economy, the problem of soil pollution is day by day serious, serious soil pollution particularly heavy metal contamination has become and has comprised at present urban soil in a denominator of interior many soil, and there have been 100,000 km in China according to estimates
2soil is contaminated, and in China's soil pollution heavy metal, As, Cd, Cr, Hg and the contour toxic heavy metal of Pb are seriously polluted.Therefore, carry out heavy metal in soil to Genotoxic Assessment, this is to soil management, ensures that the coordinated development of people ' s health, protection population resource and social economy and environment is all significant.
(3) adopted special glue method; it is the first layer frictioning; then adopt the method for traditional " sandwich " glue; at the 3rd layer, use normal fusing point that fusing point is higher instead compared with NMA glue; high being not easy of fusing point of NMA come unstuck; can finely protect colloid, therefore substantially solve the easily damaged shortcoming of traditional method offset plate.
(4) algae is the important component part of ecotope, and algae, for SCGE, is expanded to the research object of SCGE.SCGE technology is suitable for all eukaryotic cells theoretically, sample cell requirement is little, cell can be the cell of cultured cells Xi Huo somatic tissue separation, the cell of having used at present have human lymphocyte, epidermic cell, hemocyte, inoblast, testicular cell, tumour cell, animal livers cellular plant with cell and blade and yeast cell etc.But the comet of relevant algae domestic all rarely have report outward.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of step 3 Shop glue of the present invention film-making.Wherein, 1 is 1%NMA, and 2 is 0.7%LMA & algae, 3 NMA that are 1%, and 4 is frosted slide glass.
Fig. 2 is deionized water to the undamaged comet image of very thin Euglena DNA (intac nucleus-do not have the tail of a comet).
Fig. 3 is soil METAL EXTRACTION liquid to the comet image of very thin Euglena DNA damage (nucleus of damaged-have the very long tail of a comet).
Fig. 4 a and Fig. 4 b are the impact of different soils extracting solution on very thin Euglena DNA damage, wherein a is the tail momentum effects of different soils extracting solution to very thin Euglena DNA damage, b is the tail square impact of different soils extracting solution on very thin Euglena DNA damage, and * compares p<0.05 with blank.
Embodiment:
According to following embodiment, can better understand the present invention.Yet, those skilled in the art will readily understand, the described concrete material proportion of embodiment, processing condition and result thereof be only for the present invention is described, and should also can not limit the present invention described in detail in claims.
Embodiment 1:
The pre-treatment of 1 soil sample:
In the Purple Mountain, gather soil sample and contrast as cleaning soil, two local sample number of Mou Steel Plant be No. 1, certain steel and No. 2, certain steel.Sampling gathers soil sample by State Bueau of Environmental Protection's method for normalizing, and the basic physical and chemical of soil sample and heavy metal content are in Table 1.
Table 1 pedotheque basic physical and chemical and heavy metal content
1) numeric representation is mean+SD (Mean ± S.D)
After air-dry, the soil sieve that diameter is 2mm excessively, removes after plant residue, stone etc., grinds, all by 80 mesh sieves with agate mortar.Soil, after clearing up, with the warm dissolved residue of 1ml nitric acid, is then transferred to solution in 50ml volumetric flask, and constant volume, shakes up, and saves backup;
The cultivation of 2 very thin Euglenas "
Very thin Euglena is provided fresh water algae algae kind storehouse (FACHB) by typical case's culture collection council of Inst. of Hydrobiology, Chinese Academy of Sciences, adopts Checcucci(1976) culture medium culturing.Yi Mou steel mill soil extract liquid is contaminated as poisonous substance, and Purple Mountain soil extract liquid contrasts as cleaning soil, deionized water contamination as blank.Each group establish 3 parallel.Inoculum density is 0.5 * 10
5individual cell/mL left and right, culture temperature for (25 ± 1) ℃, light intensity be 80-90 μ mol/(m
2s), light dark period is 12h:12h, cultured continuously 6d.Regularly shake every day.After 6d, get well-grown cell 1mL, 3000rpm is centrifugal, after centrifugal 5min, removes liquid portion, with 1mL soil extract liquid suspension cell, obtains cell suspending liquid, moves to and in 24 orifice plates, cultivates under these conditions 3h.
3 cell preparations:
The cell of contaminating in 24 orifice plates is transferred to centrifuge tube, the centrifugal 5min of 3000rpm, centrifugal rear removal liquid portion, with PBS Eddy diffusion cell, obtains cell suspending liquid, and centrifugal purification is standby.
4 leach liquors detect the degree of injury of DNA:
4.1 paving glue:
First with 1%g/mL NMA350 μ L bottoming on frosted slide glass, then scrape off.Again 100 μ L0.7%g/mLNMA are dropped on prefabricated frosted slide glass to rapid covered, 4 ℃ of curing 10min.Then in the cell of preparation, add 0.7%g/mL LMA, resuspended algae mud, piping and druming evenly becomes cell suspension, adjusts cell concn 1 * 10
5--1 * 10
6individual cell/mL.Get 75 μ L and be added on the first layer glue, covered, 4 ℃ of curing 30min.On the LMA layer of final set, drip 100 μ L1%g/mL NMA, covered, 4 ℃ of curing 10min.
4.2 cracking:
The slide glass preparing is removed to cover glass, immerse 4 ℃ of alkaline bleach liquor cleavage liquid, cracking 20min.
4.3 electrophoresis:
Take out the slide glass after cracking, put into electrophoresis chamber, in groove, slowly inject alkaline electrophoresis liquid, after standing 20min, start electrophoresis, electric current 200mA, voltage 20V, electrophoresis time 20min.
4.4 neutralizations:
With 0.4mmol/L Tris-HCl damping fluid (pH=7.5) submergence slide glass, the each 5min of rinsing, in and 3 times;
4.5 dyeing:
Ethidium bromide (EB) the 30 μ L that drip 50 μ g/mL on glue, add a cover cover glass, microscopic examination.
4.6DNA degree of injury is analyzed:
With fluorescent microscope (BX41, Olympus), at green glow, excite lower observation, with accompanying software, under automatic exposure condition, with cold CCD, take pictures.Obtain after comet image, with CASP software analysis, measure the parameter of DNA migration: tail square (TM) and tail momentum (OTM), with it, evaluate the degree of injury of DNA.Experiment at least establish 3 parallel, get its mean value.Statistics is carried out ANOVA analysis with Origin7.5 software, and experimental group and control group are carried out to significant t-test, usings p<0.05 as significance foundation.Comet image analysis adopts CASP software, and in evaluating, tail momentum (olive tail moment, OTM) has reflected DNA content and tail of a comet shape facility in comet simultaneously, is the common counter of quantification DNA damage degree.In addition, provide tail square (TM) parameter as with reference to index, to reflect DNA damage situation more comprehensively.Every slide glass is at least analyzed 50 cells.
5. experimental result:
Fig. 2 is deionized water to the undamaged comet image of very thin Euglena DNA (intac nucleus-do not have the tail of a comet).Fig. 3 is soil METAL EXTRACTION liquid to the comet image of very thin Euglena DNA damage (nucleus of damaged-have the very long tail of a comet).Fig. 4 a and Fig. 4 b are the impact of different soils extracting solution on very thin Euglena DNA damage, wherein a is the tail momentum effects of different soils extracting solution to very thin Euglena DNA damage, b is the tail square impact of different soils extracting solution on very thin Euglena DNA damage, and * compares p<0.05 with blank.As can be seen from Figure, deionized water treatment group and cleaning soil treatment group are very little to the damage of earthworm DNA, and Nangang has compared significant difference to the damage of micro-algae DNA with control group with No. 2 soil sample treatment group No. 1.The damage that Nangang organizes micro-algae DNA for No. 2 is greater than Nangang No. 1, more consistent with heavy metal in soil content.No. 1 heavy metal in soil content of Nangang cleans the content in soil apparently higher than the Purple Mountain as shown in Table 1, and in No. 2 soil samples of Nangang, heavy metal content is significantly higher than content in No. 1 soil sample of Nangang.Therefore, with DNA tail square and tail momentum, can be used to refer to the genetic damage of heavy metal in soil to micro-algae DNA.
Described in embodiment, solution formula is as follows:
1PBS damping fluid: PBS(is without Ca
2+, Mg
2+phosphate buffered saline buffer):
Liquid 1:NaH
2pO
42H
2o0.15M(23.4g/L), add 2.34g in 100mL deionized water.
Liquid 2:Na
2hPO
412H
2o0.15M(53.72g/L), add 26.86g in 500mL deionized water.
Liquid 3:NaCl0.145M(8.5g/L), add 4.25g in 500mL deionized water.
Get 96mL liquid 1,404mL liquid 2 and 500mL liquid 3 and be mixed into 1000mL, regulating pH is 7.4.
2Checcucci(1976) nutrient solution:
CH
3cOONa3H
2o1.0g, (NH
4)
2hPO
41.0g, KH
2pO
41.0g, MgSO
47H
2o0.2g, Fe
2sO
47H
2o0.003g, CaCl
20.02g, EDTA Triplex III0.0637g; Trace element mother liquor 1mL; VITAMIN: VB
1100 μ g, VB
121 μ g; Be dissolved in 1000mL deionized water.
Trace element mother liquor: MnCl
20.00018g, CoSO
47H
2o0.0015g, ZnSO
47H
2o0.0004g, NaMoO
42H
2o0.0002g, CuSO
45H
2o0.00002g.
Nutrient solution (not adding trace element and micro-raw plain) is prepared to one night of rear placement, and 121 ℃ of high-temperature sterilization 20min then, add VITAMIN and the micro-mother liquor of filtration sterilization after cooling.
3 normal fusing point agarose gels, 0.7%, the NMA of 1%g/mL, PBS is solvent, matching while using;
Low melting-point agarose glue, the LMA of 0.7%g/mL, PBS is solvent, matching while using.
4 alkaline bleach liquor cleavage liquid
Storing solution: weigh 1.44g NaOH, 1., 3401g Na
2-EDTA, 0.012g SDS, is settled to 120mL with deionized water.Room temperature preservation.
Application liquid: get storing solution 89mL, add 1mL TritonX-100 and 10mL, and refrigeration at 4 ℃.
5 alkaline electrophoretic buffers
Weigh 42g NaOH and 1.301g Na
2-EDTA2H
2o, is dissolved in 3500mL deionized water.
6 neutral buffered liquid
Weigh 48.5gTris and be dissolved in 1000mL deionized water.With the HCl adjusting pH of 10mol/L, be 7.5.Room temperature preservation.
7 ethidium bromide staining liquid
Weigh ethidium bromide and be dissolved in deionized water, be mixed with storing solution.Lucifuge is stored.Used time is by 10 times of storing solution dilutions.Matching while using.
Claims (9)
1. detect the potential genotoxic method of heavy metal in soil pollutent, it is characterized in that: it comprises the steps:
The pre-treatment of step 1. soil sample: gather pedotheque, air-dry after, the soil sieve that to cross diameter be 2mm, removes after plant residue, stone etc., grinds, all by 80 mesh sieves with agate mortar.Soil, after clearing up, with the warm dissolved residue of 1ml nitric acid, is then transferred to solution in 50ml volumetric flask, and constant volume, shakes up, and saves backup;
Step 2. cell preparation: microalgae cell is inoculated in the soil METAL EXTRACTION liquid that step (1) obtains and is contaminated, by the microalgae cell of deionized water contamination in contrast; The above-mentioned microalgae cell that dyed poison, respectively through centrifugal removal liquid portion, with PBS Eddy diffusion cell, is obtained to cell suspension, and centrifugal purification is standby;
Step 3. paving glue:
With normal fusing point agarose (NMA) the PBS solution of 1%g/mL, (1%g/mL adds 1g NMA to step 3a. in 100mLPBS, identical to give a definition) 300--500 μ L bottoming on frosted slide glass, scrape off again, can make like this first layer glue more easily fix on slide glass;
Step 3b. prepares the first layer glue: normal fusing point agarose (NMA) the PBS solution of 60--120 μ L0.7%g/mL is dropped on the frosted slide glass that step 3a obtains to covered rapidly, 4 ℃ of curing 5-10min;
Step 3c. prepares second layer glue: to step 2, process low melting-point agarose (LMA) the PBS solution that adds 0.7%g/mL in the cell obtaining, and resuspended algae mud, piping and druming evenly becomes cell suspension, adjusts cell concn 1 * 10
5--1 * 10
6individual cell/mL.Then take out the solidifying slide of the first layer glue, gently pull out and remove lid, get 50--120 μ L and be added on the first layer glue, covered, 4 ℃ of curing 30-40min;
Step 3d. prepares the 3rd layer of glue: after the second layer solidifies, get the 3rd layer of glue of PBS solution paving of the normal fusing point agarose (NMA) of 50--120 μ L1%g/mL, after covered, put into refrigerator it is solidified;
Step 4. cracking: the slide glass that step 3d is prepared removes cover glass, immerses 4 ℃ of alkaline bleach liquor cleavage liquid (2.5mol/LNaOH, 1.0mmol/L Na
2-EDTA, 0.01%SDS(w/v), pH=10, with front adding 1%TritonX-100 and 10%DMSO), cracking 10min-2h;
Step 5. electrophoresis: take out the slide glass after cracking, put into electrophoresis chamber, slowly inject alkaline electrophoresis liquid (pH=13.0) in groove, start electrophoresis after standing 20min, electric current 200--300mA, voltage 18--25V, electrophoresis time 20min;
Step 6. neutralization: with pH7.5,0.4mmol/L Tris-HCl damping fluid submergence slide glass, the each 5min of rinsing, in and 3 times;
Step 7. dyeing: drip ethidium bromide (EB) the 30 μ L of 50 μ g/mL on glue, add a cover cover glass, microscopy in 24h;
Step 8.DNA degree of injury is analyzed: with fluorescent microscope, at green glow, excite lower observation, take pictures, obtain after comet image, with CASP software analysis, measure the parameter of DNA migration: tail square (TM) and tail momentum (OTM), with it, evaluate the potential genetoxic of heavy metal in soil.
Step 9. experiment at least establish 3 parallel, get its mean value; Statistics is carried out ANOVA analysis with Origin7.5 software, and experimental group and control group are carried out to significant t-test, usings p<0.05 as significance foundation.
2. method according to claim 1, is characterized in that: in step 1, described to clear up operating process as follows:
Take 0.2000g and cross the air-dry soil sample of 80 object in 30ml tetrafluoroethylene crucible, add 5ml HCl low-temperature heat, sample is tentatively decomposed, to be evaporated during to 2-3ml, take off slightly coldly, then add 5ml nitric acid (technique is ultrapure), 3ml hydrofluoric acid, 3ml perchloric acid, after adding a cover, in the upper temperature heating of hot plate, within about 1 hour, (notice that temperature is too not high, in order to avoid solution is emerged), then uncap, continue heating silica removal, and often shake crucible to reach the good silicon effect that flies
When being heated to emit the white cigarette of dense perchloric acid, add a cover, black organic carbon compound is fully decomposed, after black organism on crucible disappears, uncapping to drive white cigarette and steam to content is thick, depending on clear up situation can add again 2ml nitric acid,
2ml hydrofluoric acid, 2ml perchloric acid repeat said process, when white cigarette emits to the greatest extent and when content is thick again substantially, take off slightly coldly, and water rinses crucible cover and inwall.
3. method according to claim 1, is characterized in that: in step 2 and step 3, described microalgae cell is very thin Euglena cell.
4. method according to claim 1, is characterized in that: step 3a is with normal fusing point agarose 350 μ L bottoming on frosted slide glass of 1%g/mL.
5. method according to claim 1, is characterized in that: step 3b be by the normal fusing point agarose drop of 100 μ L0.7%g/mL on the prefabricated slide glass obtaining of step 3a.
6. method according to claim 1, is characterized in that: in step 3c, be to get 75 μ L cell suspensions to be added drop-wise on the first layer glue.
7. method according to claim 1, is characterized in that: in step 3d, be the normal fusing point agarose that drips 100 μ L1%g/mL on second layer glue.
8. method according to claim 1, is characterized in that: in step 4, pyrolysis time is 20min.
9. method according to claim 1, is characterized in that: in step 5, electrophoretic current is 200mA, and voltage is 20V, and electrophoresis time is 20min.
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Application publication date: 20140423 |