1. A method for rapidly detecting single cell DNA damage is characterized by comprising the following steps:

A. spreading glue

Spreading a first layer of glue, soaking the electrophoresis glass slide in 75% ethanol solution for 1-2 h, and wiping to dry; baking the glass slide on flame once before spreading the glue, dripping 0.1-0.3 mL of common agarose gel at one end of the baked surface, spreading the agarose gel to the other end by using a coating rod, and baking for later use;

laying a second layer of glue: uniformly mixing 30-50 mu L of cell suspension with 150-250 mu L of 1% low-melting-point agarose gel by blowing, dripping the mixed solution on one side of a glass slide paved with primer, slowly putting down the glass slide from the side with the primer by using a cover glass, then removing the cover glass,

B. cracking and unwinding

Dropping 100-500 mu L of lysis solution on the glass slide with the cells laid in the step A by using a surface dropping mode, rinsing the glass slide for 3 times by using sterile water, placing the glass slide in a horizontal electrophoresis tank in parallel, placing the electrophoresis tank at 4 ℃, adding electrophoresis buffer solution without loading the glass slide, soaking for 20min to enable double DNA chains to be uncoiled,

the preparation method of the cell lysate comprises the following steps: weighing 0.12-0.15 g of Tris, 0.8-1.0 g of NaOH, 14.5-15.0 g of NaCl and Na₂Putting 3.72-4.0 g of EDTA in a beaker, adding 80mL of deionized water, stirring uniformly by using a glass rod to completely dissolve the EDTA, adjusting the pH value to 10, then fixing the volume to 100mL, storing at 4 ℃ for later use, adding 100 parts of triton and dimethyl sulfoxide before use, wherein the final concentration is 1 percent,

C. electrophoresis, staining and analysis

And (3) performing basic electrophoresis at 4 ℃ and 260-320 mA constant current, after electrophoresis, sequentially performing neutralization and soaking by adopting a neutralization buffer solution and double distilled water, and then performing dyeing and fluorescence photography analysis.

2. The method for rapidly detecting single-cell DNA damage according to claim 1, wherein:

wherein, in the step A, the glass slide is a smooth glass slide with the size of 25mm multiplied by 50mm multiplied by 2mm,

when the first layer of gel is laid, the agarose gel with common melting point is preheated in a water bath kettle at 75 ℃,

after being coated evenly by a coating rod, the common agarose gel is baked by using an alcohol lamp, and can be stored for a long time after being baked, namely, the agarose gel can be taken out immediately after use.

3. The method for rapidly detecting single-cell DNA damage according to claim 1, wherein:

in the step A, when the second layer of glue is paved, the preparation method of the 1% low-melting-point agarose comprises the following steps: weighing low-melting-point agarose, adding PBS solution with the volume 100 times of the mass of the agarose, placing the agarose in a water bath kettle at 37 ℃ for dissolving,

after the cell suspension is uniformly mixed with 1 percent of low-melting point agarose gel, the mixed solution is dripped into one side of a primer glass slide in five drops,

after the cover glass is covered, the slide system is placed at 4 ℃ for 10-12min, and then the cover glass is removed by a horizontal pushing method.

4. The method for rapidly detecting single-cell DNA damage according to claim 1, wherein:

wherein, in the step C,

the preparation method of the alkaline electrophoresis buffer solution comprises the following steps: weighing Na₂0.37-0.5 g of EDTA and 12.0-15.0 g of NaOH are placed in a beaker, 800mL of deionized water is added and is stirred uniformly by a glass rod to be completely dissolved, the pH value is adjusted to 13, the volume is fixed to 1L, the mixture is stored at 4 ℃,

the preparation method of the neutralization buffer solution comprises the following steps: weighing Na₂Putting 4.85-5.0 g of EDTA in a beaker, adding 80mL of deionized water, stirring uniformly by using a glass rod to completely dissolve the EDTA, adjusting the pH value to 7.5, and then fixing the volume to 100mL, wherein the EDTA is used immediately.

5. The method for rapidly detecting single-cell DNA damage according to claim 1, wherein:

wherein in the step C, during electrophoresis, the liquid level of the electrophoresis buffer solution in the electrophoresis tank is 1-2 cm higher than that of the glass slide,

after electrophoresis, the gel was neutralized with a neutralization buffer 3 times for 5min each, and then soaked in double distilled water 3 times for 10min each.

6. The method for rapidly detecting single-cell DNA damage according to claim 1, wherein:

wherein, in the step C, the steps of dyeing and fluorescent shooting analysis are as follows:

dripping 10-20 mu L of diluted propidium iodide dye solution on a dried glass slide in a dark environment, and covering a cover glass to dye for 30min in a manner of avoiding any bubbles; editing the glass slide into a blind slide, placing the blind slide under a fluorescence microscope, selecting an objective lens with the magnification of multiplied by 20, randomly photographing each slide for 10-20 visual fields, and counting trailing cells and the trailing rate of the cells; and selecting about 60-80 trailing cells which are close to the center of the visual field, have clear staining and are dominant in trailing forms in the target sample, and measuring and calculating tail length, tail/head% DNA and tail distance through an SCGE image analysis system CASP.

7. The method for rapidly detecting single-cell DNA damage according to claim 6, wherein:

the preparation method of the propidium iodide dye solution comprises the following steps: weighing 20-25 mg of propidium iodide reagent in a brown reagent bottle, adding 10-50 mL of PBS (phosphate buffer solution) with pH7.4, placing on a vortex vibrator, fully shaking and uniformly mixing to prepare a solution with the concentration of 2mg/mL, and storing at 4 ℃ for later use; the propidium iodide working solution was used as diluted 40-fold with PBS solution.

8. The method for rapidly detecting single-cell DNA damage according to claim 1, wherein:

and C, after the neutralization operation is finished, soaking the glass slide for three times by using sterile water, sucking liquid on the glass slide by using filter paper, and drying the glass slide in a 37 ℃ thermostat, wherein the glass slide can be taken out at any time without time limitation to dye and collect a fluorescent picture.

9. A kit for rapidly detecting single cell DNA damage, which is characterized by comprising:

glass slide coated with primer, cover glass, low-melting-point agarose gel, triton 100, dimethyl sulfoxide, NaCl, Na₂EDTA, NaOH, PBS solution, Tris, propidium iodide reagent.

Background

With the accelerated development of Chinese economy, the environmental pollution is becoming more and more serious. At present, environmental pollution on a global scale is also receiving more and more attention to human health. Wherein, the damage of DNA caused by environmental pollutants can affect the genetic stability of cells and can affect offspring, thereby causing abortion, dead fetus, teratogenesis, certain hereditary diseases and the like of pregnant women. Since cellular DNA damage can cause disease and threaten human health, it is important to detect cellular DNA damage and to understand whether such damage can cause a change in a cellular event.

Since the advent of basic comet assay, various laboratories have extensively applied this technique to the detection of cellular DNA damage, and at the same time, improvements have been made to the method to enable the detection of different intracellular DNA damage, such as single and double strand breaks, base oxidative damage, DNA-DNA/DNA-protein-drug cross-links and repair.

The method comprises the steps of suspending single cells in agarose gel, carrying out electrophoresis in an electric field for a short time after cracking treatment, and then dyeing with fluorescent dye. The DNA degradation fragments are formed in the DNA damaged cells, the swimming speed in an electric field is higher, the cell nucleus presents a comet pattern, and the normal nucleus without DNA breakage keeps a spherical shape during the swimming, so that the method is a very simple and convenient way for detecting the cell damage.

In the current operation, a layer of gel is adopted for general detection of single-cell DNA damage, and the gel layer is too thin and is easy to fall off in the electrophoresis process. If the gel layer is directly thickened, the gel layer is not easy to observe due to the fact that the gel layer is too thick, and cells cannot be located on the same plane as much as possible; if a multilayer gel stacking mode is adopted, the two layers of glue surfaces cannot be well adhered, and the degumming phenomenon still exists. In addition, the currently used cell lysate contains sodium sarcosinate, which is used to destroy the phospholipid bilayer of the cell membrane, and the substance has low solubility in water, resulting in a problem that the lysate has poor solubility when it is prepared.

Disclosure of Invention

The invention aims to solve the technical problems, improves the current single-cell DNA damage detection system, provides a rapid detection method and a kit for single-cell DNA damage, and solves the problems of easy gel falling and difficult solution preparation.

The invention provides a method for rapidly detecting single cell DNA damage in a first aspect, which comprises the following steps:

A. spreading glue

Spreading a first layer of glue, soaking the electrophoresis glass slide in 75% ethanol solution for 1-2 h, and wiping to dry; and baking the glass slide on flame once before spreading the glue, dripping 0.1-0.3 mL of common agarose gel at one end of the baked surface, spreading the agarose gel to the other end by using a coating rod, and baking for later use.

In the specific operation, a smooth glass slide is selected as the glass slide, and the size of the glass slide is 25mm multiplied by 50mm multiplied by 2 mm; preheating agarose gel with a common melting point in a 75 ℃ water bath kettle before spreading the agarose gel; after the glue is spread, a coating rod is adopted to coat the common agarose gel evenly, and then the agarose gel is baked by using an alcohol lamp outer flame, and can be stored for a long time after being baked, namely the agarose gel is taken immediately and directly spread on the second layer.

Laying a second layer of glue: and (3) uniformly blowing and stirring 30-50 mu L of cell suspension and 150-250 mu L of 1% low-melting-point agarose gel, dripping the mixed solution on one side of the slide glass paved with the primer, slowly putting down the slide glass from the side with the primer by using a cover glass, and then removing the cover glass.

In particular, the preparation method of the 1% low-melting-point agarose comprises the following steps: the low melting point agarose was weighed, added with a PBS solution in a volume 100 times the mass of the agarose, and dissolved in a water bath at 37 ℃.

And (3) uniformly mixing the cell suspension and 1% low-melting-point agarose gel, dripping the mixed solution into one side paved with a primer glass slide in five drops, covering a cover glass, placing the glass slide system at 4 ℃ for 10-12min, and removing the cover glass by using a horizontal pushing method.

B. Cracking and unwinding

Dropping 100-500 mu L of lysis solution on the glass slide with the cells laid in the step A by using a surface dropping mode, rinsing the glass slide for 3 times by using sterile water, placing the glass slide in a horizontal electrophoresis tank in parallel, placing the electrophoresis tank at 4 ℃, adding electrophoresis buffer solution without loading the glass slide, soaking for 20min to enable double DNA chains to be uncoiled,

the preparation method of the cell lysate comprises the following steps: weighing 0.12-0.15 g of Tris, 0.8-1.0 g of NaOH, 14.5-15.0 g of NaCl14, and Na₂Putting 3.72-4.0 g of EDTA in a beaker, adding 80mL of deionized water, stirring uniformly by using a glass rod to completely dissolve the EDTA, adjusting the pH value to 10, then fixing the volume to 100mL, storing at 4 ℃ for later use, adding 100 parts of triton and dimethyl sulfoxide before use, wherein the final concentration is 1%.

Through experiments, when the cell lysate is prepared, sodium sarcosine commonly used for destroying phospholipid bilayers of cell membranes is saved, so that the damage of the lysate to the cell membranes is kept, and the problem of poor dissolution when the lysate is prepared is solved.

C. Electrophoresis, staining and analysis

And (3) performing basic electrophoresis at 4 ℃ and 260-320 mA constant current, after electrophoresis, sequentially performing neutralization and soaking by adopting a neutralization buffer solution and double distilled water, and then performing dyeing and fluorescence photography analysis.

The preparation method of the alkaline electrophoresis buffer solution comprises the following steps: weighing Na₂0.37-0.5 g EDTA and 12.0-15.0 g NaHO are put into a beaker, and 8 is added00mL of deionized water is stirred by a glass rod to be completely dissolved, the pH value is adjusted to 13, then the volume is adjusted to 1L, and the mixture is stored at 4 ℃.

The preparation method of the neutralization buffer solution comprises the following steps: weighing Na₂Putting 4.85-5.0 g of EDTA in a beaker, adding 80mL of deionized water, stirring uniformly by using a glass rod to completely dissolve the EDTA, adjusting the pH value to 7.5, and then fixing the volume to 100mL, wherein the EDTA is used immediately.

During electrophoresis, the liquid level of an electrophoresis buffer solution in an electrophoresis tank is 1-2 cm higher than that of a glass slide; after electrophoresis, the gel was neutralized with a neutralization buffer 3 times for 5min each, and then soaked in double distilled water 3 times for 10min each. After the glass slide is soaked in sterile water for three times, liquid on the glass slide is sucked dry by using filter paper and then is dried in a 37 ℃ thermostat, and the glass slide can be taken out at any time without time limitation to dye and collect a fluorescent picture.

The steps for performing the staining and the fluorescent photography analysis were as follows: dripping 10-20 mu L of diluted propidium iodide dye solution on a dried glass slide in a dark environment, and covering a cover glass to dye for 30min in a manner of avoiding any bubbles; editing the glass slide into a blind slide, placing the blind slide under a fluorescence microscope, selecting an objective lens with the magnification of multiplied by 20, randomly photographing each slide for 10-20 visual fields, and counting trailing cells and the trailing rate of the cells; and selecting about 60-80 trailing cells which are close to the center of the visual field, have clear staining and are dominant in trailing forms in the target sample, and measuring and calculating tail length, tail/head% DNA and tail distance through an SCGE image analysis system CASP.

The preparation method of the propidium iodide dye solution comprises the following steps: weighing 20-25 mg of propidium iodide reagent in a brown reagent bottle, adding 10-50 mL of PBS (phosphate buffer solution) with pH7.4, placing on a vortex vibrator, fully shaking and uniformly mixing to prepare a solution with the concentration of 2mg/mL, and storing at 4 ℃ for later use; the propidium iodide working solution was used as diluted 40-fold with PBS solution.

In a second aspect of the invention, a rapid detection kit for single-cell DNA damage is provided, which comprises a glass slide coated with a primer, a cover glass, a low-melting-point agarose gel, triton 100, dimethyl sulfoxide, NaCl, Na₂EDTA, NaOH, PBS solution, Tris, propidium iodide reagent. Selecting required reagent from single cell DNA damageAnd (5) solution preparation is carried out.

Effect of the invention

Firstly, adopting a two-layer glue method when spreading glue, baking a glass slide on flame once before spreading glue when spreading a first layer, dripping common agarose gel on one end of a baked surface, spreading the agarose gel to the other end by using a coating rod, baking for later use, and then spreading glue on a second layer according to the normal steps. The two-layer glue method can ensure that the glue surface is well attached, and cells are positioned on the same plane as much as possible, so that the effect is better, meanwhile, after the first layer of glue is baked, the first layer of glue can be stored for a long time, namely, the glue can be taken immediately, and the second layer of glue spreading can be directly carried out, so that the glue spreading method is simple, convenient and easy.

Secondly, when the cell lysate is prepared, sodium sarcosinate which is commonly used for destroying phospholipid bilayers of cell membranes is saved, so that the damage of the lysate to the cell membranes is still maintained, and the problem of poor dissolution when the lysate is prepared is solved.

Thirdly, the glass slide processed after the electrophoresis and neutralization operation can be taken immediately after use, and the glass slide after the comet electrophoresis is stored for a long time and is taken out at any time to be dyed and the fluorescence picture is collected.

Therefore, the invention provides a simpler method for detecting the damage of the single cell DNA, solves the problems of easy degumming, difficult observation, difficult operation and the like in the detection process, ensures that the detection is easier to operate, and ensures that the operation is more flexible and convenient and can be carried out at any time.

Drawings

FIG. 1 shows the comet assay results of single cell DNA damage of HepG2 cells by the method of the present invention: (A) the image is comet images of the cells after different drug dose treatments, and (B) is the comet positive proportion of the cells under different drug dose treatment conditions.

Fig. 2 is a CASP software analysis process.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

Example A method for rapidly detecting DNA damage of a single cell

1. Spreading glue

Preparing before glue making: the water bath kettle at 75 ℃ and 37 ℃ is opened half an hour before operation.

A first layer of glue: the first layer of glue is base glue, a smooth glass slide is selected, all the glass slides are cleaned, soaked in alcohol for 1 hour and then wiped dry by using a piece of lens wiping paper; and (3) lighting an alcohol lamp, baking the glass slide once on flame before spreading the gel, dripping 0.1-0.3 mL of agarose gel with a common melting point preheated in a 75 ℃ water bath kettle at one end of the baked surface, immediately spreading the gel to the other end by using a triangular glass rod, and then baking the gel by using the outer flame of the alcohol lamp for later use.

And (3) gluing a second layer: and (3) uniformly blowing and stirring 30-50 mu L of cell suspension and 150-250 mu L of 1% low-melting-point agarose gel, dripping 5 drops of the mixed solution on one side of a glass slide with a primer, slowly putting down a cover glass from the side with the primer without generating bubbles, then placing the glass slide in an environment at 4 ℃, and removing the cover glass by a horizontal pushing method after 10-12 min.

2. Cracking and unwinding

Preparing a cell lysate: weighing 0.12-0.15 g of Tris, 0.8-1.0 g of NaOH, 14.5-15.0 g of NaCl and Na₂Putting 3.72-4.0 g of EDTA in a beaker, adding 80mL of deionized water, stirring uniformly by using a glass rod to completely dissolve the EDTA, adjusting the pH value to 10, then fixing the volume to 100mL, storing at 4 ℃ for later use, adding 100 parts of triton and dimethyl sulfoxide before use, wherein the final concentration is 1%.

And (3) dripping 100-500 mu L of lysis solution into the glass slide with the cells laid thereon in a surface dripping mode, rinsing the glass slide for 3 times by using sterile water, placing the glass slide in a horizontal electrophoresis tank in parallel, placing the electrophoresis tank at 4 ℃, adding an electrophoresis buffer solution into the glass slide without loading, and soaking for 20min to ensure that the double strands of the DNA are uncoiled.

3. Electrophoresis and neutralization

Performing electrophoresis at 4 ℃ and 260-320 mA for 10 min. After electrophoresis, the gel was neutralized with a neutralization buffer for 3 times, 5min each time, and then soaked in sterile water for 3 times, 10min each time. After the liquid on the slide was blotted with filter paper, it was dried in a 37 ℃ incubator. After drying, the fluorescent picture can be taken out at any time without being limited by time to be dyed and collected.

The preparation method of the alkaline electrophoresis buffer solution comprises the following steps: weighing Na₂0.37-0.5 g of EDTA and 12.0-15.0 g of NaOH are placed in a beaker, 800mL of deionized water is added and is stirred uniformly by a glass rod to be completely dissolved, the pH value is adjusted to 13, then the volume is adjusted to 1L, and the mixture is stored at 4 ℃.

The preparation method of the neutralization buffer solution comprises the following steps: weighing Na₂Putting 4.85-5.0 g of EDTA in a beaker, adding 80mL of deionized water, stirring uniformly by using a glass rod to completely dissolve the EDTA, adjusting the pH value to 7.5, and then fixing the volume to 100mL, wherein the EDTA is used immediately.

4. Staining, fluorescence photography analysis

And (3) dripping 10-20 mu L of the diluted PI dye solution on a dried glass slide, and covering a cover glass (avoiding any bubbles) for dyeing for 30 min. All the steps are operated in a dark environment, so that the additional damage of light to DNA is avoided. Each dose group was repeated 3 times. The slide glass is compiled into a blind glass, and is placed under a fluorescence microscope (objective lens multiplied by 20 times), 10-20 visual fields (depending on the number of cells) are randomly photographed for each glass, the trailing cells are counted, and the cell trailing rate (shape index) is calculated, wherein the trailing rate is (number of trailing cells/number of observed cells) × 100%. And selecting about 60-80 trailing cells which are close to the center of the visual field, are clearly dyed and have trailing forms which are dominant in the target sample, and measuring and calculating objective distance indexes and composite indexes such as tail length, tail/head (length), tail/head% DNA, tail distance and the like through an SCGE image analysis system CASP.

Example two verification experiment

1. Test cell

HepG2 cells were selected as the subject.

2. Test cell preparation and injury management

Preparing cells in logarithmic growth phase into cell suspension, centrifuging the cells to remove pancreatin and a culture medium, and then suspending the cells again by using fresh culture medium for use; adjusting cell density to 1X 10⁵～1×10⁶cells/ml (by a cell counter technology), inoculating the cells on a 6-hole cell culture plate, detecting the cells by a rear light mirror for 24 hours to confirm that the adherent growth is normal,the medium in the wells was discarded and the drug-infected cells were prepared and cultured.

After the mother solution of the drug was diluted with HepG2 cell culture medium to a test solution of the corresponding concentration (5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M), 2ml of the test solution was added to each well of the prepared cell culture plate, and the resulting mixture was left to stand in the corresponding incubator for the corresponding time. The control group uses 0.1% dimethyl sulfoxide as a blank control group, and each group of experiments are independently repeated three times.

3. Analysis of results

The method in the first embodiment is adopted for detection, and the degumming condition does not occur in the electrophoresis process. After the HepG2 cells are infected with drugs with different doses, comet experiments and image acquisition are carried out, and then the cells are analyzed by CASP software.

With the increase of the concentration of the drug solution, the tail moment of "comet" is gradually prolonged (fig. 1A), and the comet positive ratio of the cells is also increased with the increase of the concentration of the drug solution (fig. 1B), which indicates that the DNA damage degree of the cells is positively correlated with the concentration of the drug solution. The cell lysate can completely realize cell lysis without causing any influence on subsequent electrophoresis and dyeing.

The general process of CASP software analysis is shown in FIG. 2. Each gradient of each experimental material was repeated three times under parallel experimental conditions, and 100-150 cells were taken from each replicate slide and analyzed in the image input software to record data. The head length, tail length, head DNA content, tail DNA content, TM value and OTM value of each cell DNA comet can be automatically generated by software, wherein the tail length, the head DNA content and the TM value are indexes for analyzing cell DNA damage.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.