CN106568824A - Method for detecting DNA damage of mussels under stress of compound heavy metals - Google Patents
Method for detecting DNA damage of mussels under stress of compound heavy metals Download PDFInfo
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Abstract
The invention belongs to the field of genetic ecotoxicology research, and specifically relates to a method for detecting the DNA damage of mussels under the stress of compound heavy metals. The method comprises the following steps: culturing mussels, extracting a blood corpuscle sample, measuring the survival rate of cells, carrying out compound in-vitro exposure, and measuring the DNA damage, wherein in the step of compound in-vitro exposure, one or more of a Cu solution, a Cd solution, and a Pb solution is added, the solutions are evenly mixed, and mussels are exposed to the heavy metal solution at a temperature of 4 DEG C for 60 minutes in the absence of light. The DNA damage measuring step comprises a centrifugal treatment, cell embedding, cell disruption, electrophoresis, neutralization, observation and counting, and data analysis. The provided method can precisely represent the effect of pollution caused by multiple heavy metals on the damage of DNA of mussels.
Description
Technical field
The invention belongs to ecological matrix is because of toxicologic study field, and in particular to Compound Heavy Metals stress under a kind of conditions in vitro
The detection method of caused mussel hemocyte DNA damage.
Background technology
With the quickening of industrialization and urbanization process, heavy metal pollution problem is increasingly serious, and the heavy metal in environment is led to
Cross the processes such as river, rainfall to will transfer in marine environment, these environment heavy metals are by food chain and food web in organism
Interior accumulation is simultaneously persistently present, and serious threat is brought to biological and ecological environment.Mytilus eduliss（Mytilus galloprovincialis）It is the modal economic shellfish in China East China Sea, because it has widely distributed, anchorage, filter
Feeding habits and it is accumulative the characteristics of, be widely used in toxicologic study.Using mussel as model organism, using experiment in vitro
Method, the DNA damage level of heavy metal level determination mussel hemocyte in simulated environment, so as to judge that heavy metal is given birth to Yu Haiyang
The stress pressure of thing.Determining for DNA damage can be a kind of existing using the method for single cell gel electrophoresis, also known as comet
Individual cell level carries out the detection method of DNA damage, has the advantages that easy, quick, efficient, consumption is little.But current laboratory
The many methods using experiment in vivo of research, concentrate coercion of a certain single heavy metal of research to organism, it is impossible to enough timely
Damage of the various heavy combined pollution to biological cell in actual environment is accurately reflected, and detects time-consuming longer,
It is dfficult to apply to Site Detection.Current existing comet method can often occur agarose gel during detection and break
Broken, degumming, the poor situation of cell attachment type and resuspended dispersibility, and also it is more difficult by comet compared with the DNA fragmentation fracture of short chain
Experiment is presented, therefore the accuracy in detection using the method development of comet is somewhat limited.
The content of the invention
In order to make up the deficiencies in the prior art, the technical problem to be solved is to provide one kind and can detect compound
Under heavy metal stress in the single blood lymphocyte of mussel DNA damage method for quick, the method not only can accurately reflect
The situation of infringement of the various heavy combined pollution to mussel hemocyte DNA, can also accurately be presented that pollutant cause is single-stranded
The fracture of DNA fragmentation.Importantly, during detection, in order to meet the accuracy of detection, can effectively avoid down
Layer agarose gel broken and come off.Upper strata gel prepared by the present invention, not only can ensure that effectively with lower floor's gel
Attachment, and the hemocyte for being embedded can be good at dispersion wherein, it is ensured that individual cell level can be detected.Meanwhile, this
Technology carries out experiment in vitro using the hemocyte isolated, and the life and health of laboratory animal is not affected, when not only shortening experiment
Between, experimental cost is reduced, and the ethnics Problem that may be brought using biological sample is avoided, be conducive to animal protection.Again
It is secondary, present invention could apply to the quick blood cell samples collection in scene, detection, speed and will not be because of biological sample
Gather in a large number and affect local ecological balance.Finally, mussel hemocyte changes with higher response environment, especially pollutant
Sensitivity under stress pressure, the use of the invention can realize for marine organisms security threat caused by heavy metal institute and
The forewarning function that the marine eco-environment deteriorates, has a extensive future.
To solve above-mentioned technical problem, the present invention is comprised the following steps：
（1）Blood cell samples are extracted：Liquid of haemolymph is extracted from mussel closed shell flesh carries out centrifugal treating in centrifuge tube, and bar is centrifuged
Part：4 DEG C of temperature, 2000 ~ 10000rpm of rotating speed, 2 ~ 10min of time；Supernatant is removed after centrifugation, gained lower sediment thing is
Blood cell samples, save backup under the conditions of gained blood cell samples are placed in into -4 ~ 4 DEG C；
（2）Cell survival rate is determined：Take certain volume step（1）Middle gained blood cell samples add equal-volume in centrifuge tube
Cell re-suspension liquid, adds equal-volume 0.4% after mixing（v/v）Trypan Blue dye, stand 3 ~ 10min；Blood after dyeing is thin
Born of the same parents' sample is placed on blood counting chamber, and observation is counted, and calculates cell survival rate；If cell survival rate is higher than 90% and cell
Quantity is 107, then exposure experiment is carried out；Otherwise, then return to step（1）, blood cell samples are extracted again or cell sample is entered
Row dilution；
（3）Compound external exposure：Take certain volume step（1）Middle gained blood cell samples are added isopyknic in centrifuge tube
The mixed solution of various heavy, 60min is exposed after mix homogeneously under 4 DEG C of dark conditions；
（4）The measure of DNA damage：
A. by Jing steps（3）Middle gained blood cell samples centrifugal treating, centrifugal condition such as step（1）It is described, after centrifugation, in removal
Clear liquid is precipitated cell mass；
B. cell embedding：
It is 0.5% ~ 1.5% normal melting point agarose by concentration, after microwave heating dissolving, is coated on microscope slide, placed
It is standby after night；
By concentration be placed in 37 DEG C of water-baths after 0.5% ~ 1.0% low melting-point agarose heating fully dissolving be incubated it is standby；
With gained sedimentation cell group in phosphate buffer solution or resuspended step a of HBSS balanced salt solutions, recentrifuge, remove
Supernatant, centrifugal condition such as step（1）It is described；
By stepLow melting-point agarose after process adds stepSedimentation cell group after middle process, after mix homogeneously
Deca is in stepIn on obtained microscope slide, covered is placed under 4 DEG C of dark conditions and fixes 30 ~ 60min；
C. cell lysis：
It is prepared by storing solution：2.5M NaCl, 100mM EDTA and 10mM Tris base mixed dissolutions, add NaOH to adjust pH value
For 10, it is placed in 4 DEG C of environment and saves backup；
It is prepared by working solution：0.5ml triton x-100s and 5ml dimethyl sulfoxide are added in 50ml storing solutions, working solution is obtained final product；
Cracking process：Remove step bIn coverslip after, by microscope slide immerse color jar in working solution in, it is black at 4 DEG C
20 ~ 60min is cracked under dark condition；
D. electrophoresis：
It is prepared by electrophoresis liquid：0.3M NaOH and 1mM EDTA are dissolved in pure water, adjust pH >=13, are placed in 4 DEG C of environment and are preserved standby
With；
De-rotation process：Microscope slide after cracking in step c is positioned in Horizontal electrophoresis tank, electrophoresis liquid is added, in 4 DEG C of dark bars
0 ~ 40min of unwindase 12 under part；
Electrophoresis process:Open electrophresis apparatuses, using electric current of voltage regulation under 4 DEG C of dark conditions 20 ~ 40min of electrophoresis；
E. neutralize:
Microscope slide neutral buffer in step d after electrophoresis is rinsed repeatedly under the conditions of 4 DEG C；
F. observation is counted, data analysiss.
Further, the step（1）In centrifugal condition it is as follows：Temperature is 4 DEG C, and rotating speed is 10000rpm, and the time is
2min。
Further, the step（3）Middle heavy metal solution has three classes, and the first kind is the pure solution of Cu, Cd, Pb, Equations of The Second Kind
For Cu+Cd, Cd+Pb, Pb+Cu composite solution, the 3rd class is Cu+Cd+Pb composite solutions.
Further, the step（4）B cell embeddings：
By the normal melting point agarose that concentration is 1.5%, after microwave heating dissolving, the wherein one side of microscope slide is coated in, is put
It is standby after putting overnight；
By concentration be 0.75% low melting-point agarose heating for dissolving after be placed in 37 DEG C of water-baths be incubated it is standby；
With gained sedimentation cell group in resuspended step a of phosphate buffer solution that pH is 7.4, recentrifuge, supernatant is removed,
Centrifugal condition such as step（1）It is described；
By stepLow melting-point agarose after process adds stepSedimentation cell group after middle process, after mix homogeneously
By the 80ul low melting-point agarose Deca containing cell in stepIn be coated with the microscope slide of normal melting point agarose, cover
Coverslip, is placed under 4 DEG C of dark conditions and fixes 60min.
Further, the step（4）De-rotation process in d：Microscope slide after cracking is positioned over into water by row ordered arrangement
In flat electrophoresis tank, each column is booked, and adds electrophoresis liquid, the unwindase 12 0min under 4 DEG C of dark conditions.
Further, the step（4）Step e is as follows：By the microscope slide neutral buffer after step d gained electrophoresis
Rinse repeatedly under the conditions of 4 DEG C 3 times, each 10min.
Further, the step（1）Middle utilization liquid-transfering gun is drawn and removes supernatant.
Further, the step（4）Middle resuspension process is using the processing method of liquid-transfering gun piping and druming.
Further, the compound method of neutral buffer described in step e is：48.44g Tris base are added
In 1L water, fully pH is adjusted again after dissolving for 7.5.
Beneficial effects of the present invention：
（1）The present invention carries out experiment in vitro using hemocyte, the experiment in vivo for replacing traditional whole mussel individuality of utilization to carry out,
Can the effectively save time, reduce experimental cost, and will not to fatal damage is caused using mussel, be conducive to animal protection.
（2）The present invention increases compound external exposing step, hemocyte is exposed to respectively single heavy metal solution and is combined
In heavy metal mixed solution, the lower mussel hemocyte DNA damage of single heavy metal stress not only can be detected, can also be studied
The situation of the lower mussel hemocyte DNA damage of Compound Heavy Metals stress, can heavy-metal composite pollution shape effectively in simulating reality environment
Condition, effectively improves conventional efficient and experiment accuracy.
（3）Two-layer agarose is employed in the cell embedding step of the present invention（Lower floor is 1.5% normal melting point agarose,
Upper strata is 0.75% low melting-point agarose）, and change the preparation time of two-layer agarose gel（Prepare in advance and fresh preparation）
Be conducive to strengthening the frictional force between two-layer agarose, increase the adhesive ability between two-layer glue, finally realize good cell
Embedding effect, meanwhile, agarose gel is not broken, come off, and cell adhesiveness and degree of scatter are improved.The improvement of the technology
The use of agarose in experiment can be reduced, experimental procedure is reduced, it is time-consuming, improve Detection results.
（4）The present invention is used cooperatively using the agarose of different melting points, it is ensured that also may be used compared with the fracture of the DNA fragmentation of short chain
Presented by electrophoresis process so that gained comet image is more complete, clear, accurate, facilitates follow-up data to process, be conducive to protecting
The accuracy of card data.
（5）Experimentation to fixation, crack, untwist, the condition such as time used by series of steps such as electrophoresis, neutralization is carried out
Optimization, reduces the additional injuries that experimentation is caused to cell DNA while ensureing that DNA damage can accurately be presented, and helps
In the later stage while experiment accuracy is ensured, complete clearly image is obtained（Any one process operation is improper or condition not
Properly all it is likely to result in the failure of an experiment）.
（6）Can be used for the measure of live marine organisms blood sample after method empirical tests provided by the present invention, quickly
Accurately, and sensitivity is higher.
（7）Method provided by the present invention can apply to marine organisms security risk assessment and marine ecology safety
During monitoring and warning.
Description of the drawings
Fig. 1 is the electrophoresis fluorescence image of mussel hemocyte in the present invention；
Fig. 2 is the degree of impairment of mussel hemocyte DNA under heretofore described experiment condition.
Specific embodiment
The present invention is further described in detail with reference to specific embodiment.
The mussel adopted in this example is for Mytilus eduliss（Mytilus galloprovincialis）, concrete detecting step is such as
Under：
1) culture of mussel：Mytilus eduliss are positioned over containing artificial seawater（Salinity：30‰~33‰）Glass jar in, be placed in perseverance
Cultivate 2 weeks in warm illumination box, water is changed daily once.Condition of culture：15 DEG C, 66% illumination.
2) blood cell samples are extracted：27 or so Mytilus eduliss samples are selected, is divided into 9 groups, 3 per group, respectively from Mytilus eduliss
400ul liquid of haemolymph is extracted in closed shell flesh, 2 parts are divided into, every part of 200ul is placed in the centrifuge tube through numbering, and is used respectively
In cell survival rate and the measure of DNA damage；The liquid of haemolymph of extraction is carried out into centrifugal treating：Centrifuge is opened, temperature is dropped
It is to 4 DEG C, centrifuge tube is symmetrically placed in centrifuge, centrifugal condition：2000 ~ 10000rpm of rotating speed, 2 ~ 10min of time, preferably
2min is centrifuged under the conditions of 10000rpm, it is ensured that the shorter time reaches optimal separating effect, reduces centrifugal process thin to blood
The damage that born of the same parents' sample is produced；Centrifugation is finished, and is drawn using liquid-transfering gun and is removed supernatant, and gained lower sediment thing is hemocyte sample
Product, save backup under the conditions of gained blood cell samples are placed in into -4 ~ 4 DEG C；Removing supernatant method using liquid-transfering gun not only can
Guarantee more completely removes supernatant, can also effectively reduce the loss of cell.Liquid of haemolymph extracts syringe needle used in this step
Specification is 22G, and 1ml needle tubings of arranging in pairs or groups, syringe needle is customized in Suzhou Lingyan Crag Medical Devices Co., Ltd..
3) cell survival rate is determined：To step 2) in gained a copy of it blood cell samples in add 200ul cells it is resuspended
Liquid, is gently blown and beaten to cell using liquid-transfering gun and is uniformly dispersed.It is added thereto to equal-volume 0.4%（v/v）Trypan Blue dye, mixes
3 ~ 10min, preferred 3min are placed afterwards；The blood cell samples 50ul drops after dyeing are taken in being stamped coverslip（22×22mm）Blood cell
On counting chamber, counted under the microscope.Cell survival rate=viable count/total cellular score × 100%, in the lattice of total cellular score=5
Cell quantity/0.02（Volume in 5 lattice）× extension rate, wherein cell survival rate show that cell state is good more than 90%
It is good；Cell quantity is 107, minimum cell quantity needed for comet can be met, also avoid that cell quantity is excessive to cause cell weight
It is folded, affect observation.If cell survival rate is higher than 90% and cell quantity is 107, then into step（4）, carry out DNA damage experiment；
Otherwise, then return to step（2）, blood cell samples are extracted again or carry out diluted sample.Cell concentration is very few, when later observations are counted
It is difficult；Quantity crosses conference and causes cell overlap phenomenon to occur, and causes software identification difficult.It is resuspended cell to be carried out using liquid-transfering gun
Process can be such that cell adherent after centrifugation is uniformly resuspended in a small amount of re-suspension liquid.
4) it is combined external exposure：To step（1）200ul experimental solutions are added in middle gained blood cell samples, is sufficiently mixed
Uniformly.The experimental solutions are respectively：Phosphate buffer solution group（PBS）（Blank control group）, 1000uM H2O2Group（Positive control
Group）, Cu solution groups（100ug/l）, Cd solution groups（500ug/l）, Pb solution groups（30mg/l）, Cu+Cd composite solution groups, Cd+
Pb composite solution groups, Pb+Cu composite solution groups, Cu+Cd+Pb composite solution groups；Wherein, the concentration of experimental solutions is according to early stage
Concentraton gradient experimental result selects what is determined, adds and exposure under 4 DEG C of dark conditions is placed in after experimental solutions and mix homogeneously
60min.Early stage Concentraton gradient experiment in, Cu Concentraton gradient be 50,100,500ug/l；Cd Concentraton gradient be 150,500,
1500ug/l;Pb Concentraton gradient be 3,15,30mg/l；Selected to cause substantially anti-according to early stage Concentraton gradient experimental result
The concentration answered is combined external exposed experiment for carrying out
5) measure of DNA damage：
A. by Jing steps 4）Middle gained blood cell samples centrifugal treating, centrifugal condition such as step 2）It is described, after centrifugation, remove supernatant
Liquid obtains sedimentation cell group；
B. cell embedding：
By the normal melting point agarose that concentration is 1.5%, after microwave heating fully dissolves, wherein the one of microscope slide is coated in
Face, it is standby after standing overnight；
By concentration be 0.75% low melting-point agarose heating for dissolving after be placed in 37 DEG C of water-baths be incubated it is standby；
With the phosphate buffer solution that pH is 7.4（PBS）Gained sedimentation cell group, recentrifuge, in removal in resuspended step a
Clear liquid, centrifugal condition such as step（2）It is described；
To stepStep is added in sedimentation cell group after middle process180ul low melting-point agaroses after process, obtain carefully
Born of the same parents' agarose mixed liquor, liquid-transfering gun pipettes 80ul cell agarose mixed liquors and is transferred to stepIn obtained covered normal molten
On the microscope slide of point agarose, coverslip is gently put down from drop side during cover plate, prevent the generation of bubble, do not pressed with handss.
The microscope slide for covering coverslip is placed under 4 DEG C of dark conditions and fixes 30 ~ 60min, preferred 60min, solidify agarose.Control
The each drop of cell agarose is 80ul, if each drop cell agarose volume amount is excessive, upper strata gel thicknesses can be made excessive, causes thin
Born of the same parents are layered in gel, make troubles to later observations；If if the amount of each drop cell agarose is very few, can not in cover plate
Cover uniform, affect electrophoresis process, finally affect later observations.
Normal melting point agarose in the present invention（NMP）Optional concentration is 0.5% ~ 1.5%, low melting-point agarose concentration（LMP）
0.5% ~ 0.75% is chosen as, NMP concentration preferably 1.5%, LMP concentration preferably 0.75% in the present embodiment, so that cell attachment is good
Get well, be uniformly dispersed, can preferably be presented by comet compared with the DNA fragmentation crack conditions of short chain, while higher agarose
It is not broken during microscope slide that concentration can also cause it to remove after solidifying.
C. cell lysis：
It is prepared by storing solution：2.5M NaCl, 100mM ethylenediaminetetraacetic acid（EDTA）With 10mM trishydroxymethylaminomethane（Tris
base）Mixed dissolution, it is 10 to add NaOH to adjust pH value, is placed in 4 DEG C of environment and saves backup；
It is prepared by working solution：0.5ml triton x-100s and 5ml dimethyl sulfoxide are added in 50ml storing solutions（DMSO）, obtain final product work
Liquid；
Cracking process：Step bMicroscope slide after middle gained is fixed is removed in the working solution immersed in color jar after coverslip,
20 ~ 60min is cracked under 4 DEG C of dark conditions, the pyrolysis time of preferred 60min, 60min both can ensure that mussel cell fully split
Solution, while the background value of DNA Damage will not be increased（Reference value）.
D. electrophoresis：
It is prepared by electrophoresis liquid：0.3M NaOH and 1mM EDTA are dissolved in pure water, adjust pH >=13, are placed in 4 DEG C of environment and are preserved standby
With；
De-rotation process：Microscope slide after cracking is positioned in Horizontal electrophoresis tank by row ordered arrangement, each column is booked, and avoids inclining
Tiltedly, it is ensured that sample can prevent the movement of slide position in electrophoresis process according to specific direction electrophoresis；Addition electrophoresis liquid, 4
The min of unwindase 12 0 ~ 40, preferred 20min under DEG C dark condition；20min can ensure that in mussel cell that DNA double chain is untwisted and become
It is single-stranded.
Electrophoresis process:Electrophresis apparatuses are opened, using electric current of voltage regulation（25 V）The min of electrophoresis 20 ~ 40 under 4 DEG C of dark conditions, preferably
20min；
E. neutralize:
After electrophoresis is finished, 3 times will be repeatedly rinsed under the conditions of 4 DEG C in microscope slide immersion neutral buffer in step d, every time
10min；Wherein, the compound method of neutral buffer is：48.44g Tris base are added in 1L water, fully after dissolving
PH is adjusted again for 7.5.
F. observation is counted, data analysiss：Use DNA stains（EB, 20ug/ml）In fluorescence microscopy Microscopic observation after dyeing,
Amplification generally using 100 ×, 200 × or 400 ×, it is ensured that every time observation amplification used is identical, different amplification
Presentation to image detail is variant, and this species diversity can affect the identification of later stage software, and then affect the accuracy of data.It is micro-
Observation avoids the image at agarose edge.100 cells of each microscope slide meter, both sides agarose is each 50.Adhesive plaster is used before counting
Labelling is covered, prevents anthropic factor from producing impact.Statistical analysis are carried out after counting.
Step 2 in the present embodiment）With step 4）In all resuspension process using liquid-transfering gun piping and druming method, after centrifugation
Cell mass is less, and in the attachment of centrifugation tube wall relatively closely, agents useful for same amount is again fewer when resuspended, using liquid-transfering gun rather than agitator
Or vortex vortex mixer is mixed, it can be made to split away off from tube wall, while ensureing the mix homogeneously in a small amount of solution.
Step d of the present invention is carried out under the conditions of 4 DEG C, and low temperature environment can reduce the DNA damage that operating process is caused, together
The repeatability of Shi Zengjia experimental results.
Cell re-suspension liquid is bought from green skies Bioisystech Co., Ltd in the present invention, and production code member is C0011-2；This
Bright middle HBSS balanced salt solutions select Hank ' s Balanced Salt Solution（Hank's balanced salt solution）, purchase is certainly
Sigma-Aldrich companies, production code member is 1002039047.
Electrophoretic image in the present embodiment is as shown in figure 1, be respectively from left to right the ascending cell of degree of injury, mussel
The damage results of middle DNA cells（400×）As shown in Fig. 2 using afterbody DNA percentage ratios（% Tail DNA）Refer to as analysis
Mark, wherein, Con represents blank control group；P.C. positive control is represented；Cu, Cd and Pb represent it individually to the damage of mussel DNA
Effect；Cu+Cd, Cd+Pb, Pb+Cu and Cu+Cd+Pb represent that it is acted on the complex injury of mussel DNA respectively.From Figure 2 it can be seen that
The present invention not only can be detected to the lower mussel DNA damage of single heavy metal stress, under can also studying Compound Heavy Metals stress
The situation of mussel DNA damage.The presence of heavy metal can cause the mono- chain breaks of DNA, and the degree of impairment that different heavy metals are caused is not
Together.In addition to Pb, the degree of injury caused under other Compound Heavy Metals exposure conditions is above the effect of independent heavy metal, but is not
It is multiplied.Compound Heavy Metals are illustrated to the coercion of mussel DNA and is not equal under several heavy metal independent roles to cell
Damage sum, the present invention can combined pollution situation of the heavy metal to mussel in more efficient simulating reality environment, with more existing
Sincere justice.Research Compound Heavy Metals, to mussel DNA damage situation, are that later stage Compound Heavy Metals are studied the toxic mechanism of organism
There is provided data to support.
Claims (9)
1. Compound Heavy Metals coerce the detection method of lower mussel DNA damage, it is characterised in that comprise the following steps：
（1）Blood cell samples are extracted：Liquid of haemolymph is extracted from mussel closed shell flesh carries out centrifugal treating in centrifuge tube, and bar is centrifuged
Part：4 DEG C of temperature, 2000 ~ 10000rpm of rotating speed, 2 ~ 10min of time；Supernatant is removed after centrifugation, gained lower sediment thing is
Blood cell samples, save backup under the conditions of gained blood cell samples are placed in into -4 ~ 4 DEG C；
（2）Cell survival rate is determined：Take certain volume step（1）Middle gained blood cell samples add equal-volume in centrifuge tube
Cell re-suspension liquid, adds equal-volume 0.4% after mixing（v/v）Trypan Blue dye, stand 3 ~ 10min；Blood after dyeing is thin
Born of the same parents' sample is placed on blood counting chamber, and observation is counted, and calculates cell survival rate；If cell survival rate is higher than 90% and cell
Quantity is 107, then exposure experiment is carried out；Otherwise, then return to step（1）, blood cell samples are extracted again or cell sample is entered
Row dilution；
（3）Compound external exposure：Take certain volume step（1）Middle gained blood cell samples are added isopyknic in centrifuge tube
The mixed solution of various heavy, 60min is exposed after mix homogeneously under 4 DEG C of dark conditions；
（4）The measure of DNA damage：
A. by Jing steps（3）Middle gained blood cell samples centrifugal treating, centrifugal condition such as step（1）It is described, after centrifugation, in removal
Clear liquid is precipitated cell mass；
B. cell embedding：
It is 0.5% ~ 1.5% normal melting point agarose by concentration, after microwave heating dissolving, is coated on microscope slide, stands overnight
It is standby afterwards；
By concentration be placed in 37 DEG C of water-baths after 0.5% ~ 1.0% low melting-point agarose heating fully dissolving be incubated it is standby；
With gained sedimentation cell group, recentrifuge, in removal in phosphate buffer solution or resuspended step a of HBSS balanced salt solutions
Clear liquid, centrifugal condition such as step（1）It is described；
By stepLow melting-point agarose after process adds stepSedimentation cell group after middle process, drips after mix homogeneously
It is added in stepIn on obtained microscope slide, covered is placed under 4 DEG C of dark conditions and fixes 30 ~ 60min；
C. cell lysis：
It is prepared by storing solution：2.5M NaCl, 100mM EDTA and 10mM Tris base mixed dissolutions, add NaOH to adjust pH value
For 10, it is placed in 4 DEG C of environment and saves backup；
It is prepared by working solution：0.5ml triton x-100s and 5ml dimethyl sulfoxide are added in 50ml storing solutions, working solution is obtained final product；
Cracking process：Remove step bIn coverslip after, by microscope slide immerse color jar in working solution in, in 4 DEG C of dark
Under the conditions of crack 20 ~ 60min；
D. electrophoresis：
It is prepared by electrophoresis liquid：0.3M NaOH and 1mM EDTA are dissolved in pure water, adjust pH >=13, are placed in 4 DEG C of environment and are preserved standby
With；
De-rotation process：Microscope slide after cracking in step c is positioned in Horizontal electrophoresis tank, electrophoresis liquid is added, in 4 DEG C of dark bars
0 ~ 40min of unwindase 12 under part；
Electrophoresis process:Open electrophresis apparatuses, using electric current of voltage regulation under 4 DEG C of dark conditions 20 ~ 40min of electrophoresis；
E. neutralize:
Microscope slide neutral buffer in step d after electrophoresis is rinsed repeatedly under the conditions of 4 DEG C；
F. observation is counted, data analysiss.
2. Compound Heavy Metals according to claim 1 coerce the detection method of lower mussel DNA damage, it is characterised in that：Institute
State step（1）In centrifugal condition it is as follows：Temperature is 4 DEG C, and rotating speed is 10000rpm, and the time is 2min.
3. Compound Heavy Metals according to claim 1 coerce the detection method of lower mussel DNA damage, it is characterised in that：Institute
State step（3）Middle heavy metal solution has three classes, and the first kind is the pure solution of Cu, Cd, Pb, and Equations of The Second Kind is Cu+Cd, Cd+Pb, Pb+Cu
Composite solution, the 3rd class is Cu+Cd+Pb composite solutions.
4. Compound Heavy Metals according to claim 1 coerce the detection method of lower mussel DNA damage, it is characterised in that：Institute
State step（4）B cell embeddings：
By the normal melting point agarose that concentration is 1.5%, after microwave heating dissolving, the wherein one side of microscope slide is coated in, is placed
It is standby after overnight；
By concentration be 0.75% low melting-point agarose heating for dissolving after be placed in 37 DEG C of water-baths be incubated it is standby；
With gained sedimentation cell group in resuspended step a of phosphate buffer solution that pH is 7.4, recentrifuge, supernatant is removed, from
Heart condition such as step（1）It is described；
By stepLow melting-point agarose after process adds stepSedimentation cell group after middle process, will after mix homogeneously
80ul low melting-point agarose Deca containing cell is in stepIn be coated with the microscope slide of normal melting point agarose, close the lid
Slide, is placed under 4 DEG C of dark conditions and fixes 60min.
5. Compound Heavy Metals according to claim 1 coerce the detection method of lower mussel DNA damage, it is characterised in that：Institute
State step（4）De-rotation process in d：Microscope slide after cracking is positioned in Horizontal electrophoresis tank by row ordered arrangement, each column is booked,
Add electrophoresis liquid, the unwindase 12 0min under 4 DEG C of dark conditions.
6. Compound Heavy Metals according to claim 1 coerce the detection method of lower mussel DNA damage, it is characterised in that：Institute
State step（4）Step e is as follows：Microscope slide neutral buffer after step d gained electrophoresis is rinsed repeatedly under the conditions of 4 DEG C
3 times, each 10min.
7. Compound Heavy Metals according to claim 1 coerce the detection method of lower mussel DNA damage, it is characterised in that：Institute
State step（1）Middle utilization liquid-transfering gun is drawn and removes supernatant.
8. Compound Heavy Metals according to claim 1 coerce the detection method of lower mussel DNA damage, it is characterised in that：Institute
State step（4）Middle resuspension process is using the processing method of liquid-transfering gun piping and druming.
9. Compound Heavy Metals according to claim 1 coerce the detection method of lower mussel DNA damage, it is characterised in that：Step
The compound method of neutral buffer is described in rapid e：48.44g Tris base are added in 1L water, fully after dissolving again
It is 7.5 to adjust pH.
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