CN115326519A - Sperm DNA fragment staining kit and application thereof - Google Patents
Sperm DNA fragment staining kit and application thereof Download PDFInfo
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- G—PHYSICS
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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Abstract
The invention discloses a sperm DNA fragment staining kit and application thereof. The sperm DNA fragment staining kit comprises phosphate buffer solution, rie's-Giemsa staining solution, staining solution A, staining solution B, a coating glass slide, sample diluent and a cover glass. The staining kit provided by the invention detects the DNA fragment degree of the sperm by using a sperm chromatin diffusion test principle, so that the result reliability, the sperm staining quality and other aspects are greatly improved; the staining result image is clear, the formed halo is clear and is obviously separated from the head, the sperm DNA damage degree can be conveniently judged, the detection time is short, the operation is convenient, the sperm quality analyzer can be compatible with a full-automatic sperm quality analyzer, the sperm quality analyzer can be used for large-scale laboratory detection after the automation is completed, and the efficiency can be greatly improved.
Description
Technical Field
The invention relates to a sperm DNA fragment staining kit (SCD staining method) and application thereof, belonging to the technical field of sperm detection.
Background
There are several methods reported to test normality of sperm chromatin and DNA. These methods all use dyes that bind to histones (aniline blue) or to nucleic acids (acridine orange, chromomycin) and are detected using histological methods or flow cytometry. Newer methods include the detection of DNA strand breaks, such as deoxyribonucleotide terminal transferase (TdT) -mediated deoxyribonucleotide triphosphate (dUTP) nick end labeling [ short with TUNEL (in situ end labeling, ISEL), comet assay or chromatin dispersion (SCD) assay. The results of these tests correlate with each other (Chohan et al, 2006) and correlate with sperm morphology, sperm motility and sperm survival. These tests may give additional information about the fertilization rate of standard in vitro fertilization and possibly also about the natural pregnancy rate. The sperm chromatin structure test (SCSA) is able to predict fertilization failure in vivo and in vitro (Evenson & Wixon, 2006).
Compared with other detection methods, the Sperm Chromatin Diffusion (SCD) test is simple and rapid, has reliable result and low analysis cost, does not need special instruments and reagents, has wide application range and meets the detection requirements of small and medium-sized laboratories. Some sperm DNA fragment kits (SCD method) have poor smear effect when applied to sperm DNA fragment staining. The defects are that the sperm halo is not obvious, the dyeing boundary between the halo and the sperm head is not clear, the sperm tail is not obvious in color development (non-sperm cells cannot be distinguished), and great adverse effects are brought to detection operation.
Disclosure of Invention
The invention aims to provide a sperm DNA fragment staining solution (SCD method) with obvious sperm halo, clear dyeing boundary between the halo and the sperm head, better sperm tail color development effect, higher accuracy and shorter staining time and an application staining method thereof.
In order to achieve the purpose, the invention provides a sperm DNA fragment staining kit, which comprises phosphate buffer solution, rui-Giemsa staining solution, staining solution A, staining solution B, a coating glass slide, sample diluent and a cover glass;
the pH value of the phosphate buffer solution is 6.0-7.0, wherein the content of phosphate is 0.02-0.2 mol/L;
the dyeing liquor of Rui's-Giemsa is methanol solution containing 0.1-0.5 wt% of Rui's pigment and 0.03-0.15 wt% of Jiemsa pigment;
the staining solution A is an aqueous solution containing 0.05 to 0.2 weight percent of sodium chloride, 1.0 to 10 weight percent of glycine and 0.2 to 2 weight percent of glacial acetic acid;
the staining solution B is an aqueous solution containing 0.3-3 wt% of sodium lauroyl sarcosinate, 1-10 wt% of dithio-DL-threitol, 2-10 wt% of trihydroxymethyl aminomethane, 0.2-2 wt% of disodium ethylenediamine tetraacetic acid dihydrate and 0.2-2 wt% of Tween 20, and the pH value of the aqueous solution is 7.0-8.0;
the sample diluent is 0.5-2 wt% of low-melting point agarose aqueous solution; the low-melting point agarose can be gelatinized at about 30 deg.C and melted at about 65 deg.C.
Preferably, the phosphate buffer solution further comprises a preservative ProClin 300, and the effective content of the preservative ProClin 300 is 2-5 wt%.
Preferably, the coated slide is a slide coated with 0.5 to 2wt% of an aqueous agarose solution.
More preferably, the pH value of the phosphate buffer solution is 6.4-6.8, wherein the content of phosphate is 0.06mol/L;
the dyeing liquor of Ruhry-Giemsa is methanol solution containing 0.2wt% of Ruhry pigment and 0.06wt% of Jiemsa pigment;
the staining solution A is an aqueous solution containing 0.1wt% of sodium chloride, 1.1wt% of glycine and 0.52wt% of glacial acetic acid;
the staining solution B is an aqueous solution containing 0.6wt% of sodium lauroyl sarcosinate, 3wt% of dithio-DL-threitol, 4.8wt% of trihydroxymethyl aminomethane, 0.28wt% of disodium edetate dihydrate and 0.32wt% of Tween 20, and the pH value is 7.4-7.6;
the sample diluent is a 0.8wt% aqueous low melting point agarose solution.
The invention also provides application of the sperm DNA fragment staining kit, including application in sperm DNA fragmentation degree detection for non-diagnostic purposes and/or application in sperm quality evaluation.
The invention also provides a sperm DNA fragment staining method for non-diagnostic purposes, which adopts the sperm DNA fragment staining kit as claimed in the claim to stain, and comprises the following steps:
step 1: incubating the sample dilution until it is completely dissolved, and then transferring it to 37 ℃ for equilibration for at least 5 minutes;
and 2, step: regulating the concentration of sperm (fresh sperm or thawed frozen sperm) to 5-10X 10 with physiological saline 6 Adding a certain amount (such as 60 mu L) into the sample diluent obtained in the step 1, fully and uniformly mixing, and incubating at 37 ℃ for later use;
and 3, step 3: adding a certain amount of the sperm diluent obtained in the step (2) on a pre-refrigerated coating glass slide, immediately covering a cover glass (quickly operating, not pressing the cover glass), and refrigerating (standing at 2-8 ℃ for 5 min) to completely solidify the sperm diluent;
and 4, step 4: removing the cover glass in a side-drawing mode, wherein the cover glass always clings to the glass slide to slide in the removing process, and the cover glass cannot be lifted upwards;
and 5: dripping staining solution A at room temperature (20-30 ℃) for reaction for 7 minutes, discarding the staining solution A, dripping staining solution B (1-2 mL) for reaction for 15 minutes, and discarding the staining solution B;
step 6: horizontally placing the glass slide in purified water for 5 minutes, and changing water for 1-2 times;
and 7: placing the glass slides in 70% ethanol, 90% ethanol and absolute ethanol in sequence respectively, dehydrating for 2 minutes respectively, taking out, and naturally drying in air;
and 8: mixing the Ruhry-Giemsa staining solution and phosphate buffer solution in proportion according to the number of the slides to be stained to obtain mixed staining solution, dripping the mixed staining solution on each slide to completely cover the slide, after staining for 15 minutes, washing the dye from one side of the slide by using purified water, naturally drying or blow-drying in a fume hood, and observing the staining result of sperms by using a microscope.
Preferably, the incubation temperature in step 1 is 80 ℃ and the incubation time is 20 minutes.
Preferably, the dropping amount of the staining solution A and the staining solution B in the step 5 is 1 to 2mL.
Preferably, the dyeing liquor of giemsa rapi and the phosphate buffer solution in the step 8 are mixed according to the proportion of 1.
Compared with the prior art, the invention has the beneficial effects that:
the staining kit provided by the invention detects the DNA fragment degree of the sperm by using a sperm chromatin diffusion test principle, so that the reliability of results, the staining quality of the sperm and the like are greatly improved; the kit contains the staining solution B, the sodium lauroyl sarcosine in the staining solution B can dissolve protein in sperms, but does not affect external cell membranes, and the tail characteristics of the sperms can be kept after the staining solution B is treated, so that a staining result image is clear, formed halos are clear, the boundaries with the heads are obvious, the DNA damage degree of the sperms can be conveniently judged, the detection time is short, the operation is convenient, the kit can be compatible with a full-automatic sperm quality analyzer, the kit can be used for large-scale laboratory detection after automation is completed, and the efficiency can be greatly improved.
Drawings
FIG. 1 is a diagram showing the effect of the sperm DNA fragment staining kit of the present invention in staining.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.
Examples
A sperm DNA fragment staining kit (SCD staining method) comprises phosphate buffer solution, rui's-Giemsa staining solution, staining solution A, staining solution B, coating glass slide, sample diluent and cover glass.
The pH value of the phosphate buffer solution is 6.4-6.8, wherein the content of phosphate is 0.06mol/L. The phosphate buffer solution also comprises a preservative ProClin 300, and the effective content of the preservative ProClin 300 is 2.16%.
The dyeing liquid of the Ruhry-Giemsa is methanol solution containing 0.2 percent of Reye pigment and 0.06 percent of Jiemsa pigment;
the staining solution A is an aqueous solution containing 0.1% of sodium chloride, 1.1% of glycine and 0.52% of glacial acetic acid;
the staining solution B is an aqueous solution containing 0.6% of sodium lauroyl sarcosinate, 3% of dithio-DL-threitol, 4.8% of trihydroxymethyl aminomethane, 0.28% of disodium edetate and 0.32% of Tween 20, and the pH value of the aqueous solution is 7.4-7.6;
the coating glass slide is a glass slide coated with a 1% agarose aqueous solution;
the sample diluent is an aqueous solution of agarose with a low gel temperature of 0.8%;
the preparation method of the phosphate buffer solution comprises the following steps:
1) 600-800 mL of purified water was placed in a 1000mL flask, and 6.424g of disodium hydrogen phosphate dodecahydrate and 6.64g of potassium dihydrogen phosphate were weighed, added to the flask, and stirred to completely dissolve them.
2) The pH value of the solution is detected by using a calibrated pH meter, the solution is qualified from 6.4 to 6.8, the partial acid is adjusted by using concentrated disodium hydrogen phosphate, and the partial alkali is adjusted by using concentrated potassium dihydrogen phosphate.
3) 7.2mL of ProClin 300 was measured and added to the flask, and the mixture was stirred to dissolve completely.
4) The volume is 1000mL with purified water.
The preparation method of the dyeing liquid of Rui's-Giemsa comprises the following steps:
1) 2g of Reynold's pigment and 0.6g of Giemsa pigment are weighed into a grinder, and 1000mL of methanol is added. Grinding for 5 minutes, waiting for 1 minute, grinding for 5 minutes again, waiting for 1 minute, and grinding for 5 minutes again;
2) Qualitative filter paper was placed in a 120mm buchner funnel, connected to a 1000mL suction flask and a circulating water multipurpose vacuum pump. Repeatedly filtering the liquid obtained in the step 1) for 3 times.
3) The volume is adjusted to 1000mL by methanol, and the solution is transferred to a brown bottle for storage.
The preparation method of the staining solution A comprises the following steps:
1) 500 to 600mL of purified water was placed in a 1000mL beaker, and 1g of sodium chloride, 11g of glycine and 5.2mL of glacial acetic acid were weighed, added to the 1000mL beaker, and stirred to dissolve them.
2) Transferring into a constant volume bottle, adding pure water to a constant volume of 1000mL, and turning upside down and mixing.
The preparation method of the staining solution B comprises the following steps:
1) Taking 600-700 mL of pure water, placing the pure water in a 1000mL beaker, weighing 6g of sodium lauroyl sarcosinate, 30g of dithio-DL-threitol, 48g of trihydroxymethyl aminomethane and 2.8g of disodium ethylenediamine tetraacetic acid dihydrate, adding the mixture into the beaker, stirring the mixture to dissolve the mixture, and completely transparent solution. Then, 3.2mL of Tween 20 was measured and added to the beaker, and the mixture was stirred to dissolve.
2) Under the monitoring of a PH meter, hydrochloric acid is slowly added to adjust the PH value to 7.4-7.6.
3) Transferring into a holding bottle, adding pure water to a constant volume of 1000mL, and turning upside down to mix evenly.
The preparation method of the coating glass slide comprises the following steps:
1) Weighing 1g of agarose, placing the agarose in a container capable of containing 100mL of agarose, adding 50mL of purified water, stirring, and placing the agarose in a microwave oven to heat for 1-2 minutes on high fire to completely dissolve the agarose.
2) Adding purified water preheated to 70 ℃ to a constant volume of 100mL, stirring and mixing uniformly, and preserving heat in a water bath at 60 ℃.
3) Coating the solution in the step 2) on the surface of the glass slide once when the solution is hot by using a hairbrush, and putting the glass slide into an oven to be dried at 80 ℃.
The preparation method of the sample diluent comprises the following steps:
1) Weighing 0.8g of agarose with low gel temperature, adding the agarose into a 100mL glass bottle, adding 50mL of purified water, stirring, and then placing the mixture into a microwave oven to heat for 1-2 minutes on high fire, so that the agarose with low gel temperature is completely dissolved.
2) Adding purified water preheated to 70 ℃ to a constant volume of 100mL, and uniformly stirring.
3) The solution of step 2) was portioned while hot into 0.5mL LEP tubes, 0.12mL per tube.
A staining method using the sperm DNA fragment staining kit (SCD method) comprises the following steps:
1. the sample diluent vials were incubated at 80 ℃ for 20 minutes and, after complete dissolution, were then transferred to 37 ℃ for equilibration for at least 5 minutes for use.
2. Regulating sperm concentration to 5-10X 10 with physiological saline 6 and/mL, taking 60ul of the solution, adding the solution into the dissolved sample diluent, fully mixing the solution, and incubating the solution at 37 ℃ for later use.
3. 30ul of the sperm diluent prepared above was added to a previously refrigerated coated slide, immediately covered with a cover glass (quick operation, without pressing the cover glass), and left at 2 to 8 ℃ for 5 minutes to completely coagulate it.
4. Carefully remove the coverslip in a "side draw" (the coverslip slides against the slide at all times during movement, and cannot be lifted up).
5. 1-2 mL of staining solution A is dripped under the condition of room temperature (20-30 ℃) for reaction for 7 minutes, and the staining solution A is discarded. Then 1-2 mL of staining solution B is added dropwise for reaction for 15 minutes, and the staining solution B is discarded.
6. The slides were placed horizontally in purified water for 5 minutes, during which time the water was changed 1-2 times.
7. And (3) placing the glass slide into 70% ethanol, 90% ethanol and absolute ethanol in sequence, dehydrating for 2 minutes, taking out, and naturally drying in air.
8. According to the number of the slides to be dyed actually, taking a proper amount of the Rie-Giemsa staining solution and mixing the Rue-Giemsa staining solution with the phosphate buffer solution according to a ratio of 1. After dyeing for 15 minutes, the dye is not directly discarded, and the dye is slowly washed from one side of the slide by purified water and is naturally dried or blown in a fume hood.
9. The staining effect of the sperm was observed under normal light using 20-fold or 40-fold objective lens.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way and substantially, it should be noted that those skilled in the art may make several modifications and additions without departing from the scope of the present invention, which should also be construed as a protection scope of the present invention.
Claims (8)
1. A sperm DNA fragment staining kit is characterized by comprising phosphate buffer solution, rie's-Giemsa staining solution, staining solution A, staining solution B, a coating glass slide, sample diluent and a cover glass;
the pH value of the phosphate buffer solution is 6.0-7.0, wherein the content of phosphate is 0.02-0.2 mol/L;
the dyeing liquid of the Ruhry-Giemsa is methanol solution containing 0.1 to 0.5 weight percent of Ruhry pigment and 0.03 to 0.15 weight percent of Jiemsa pigment;
the staining solution A is an aqueous solution containing 0.05 to 0.2 weight percent of sodium chloride, 1.0 to 10 weight percent of glycine and 0.2 to 2 weight percent of glacial acetic acid;
the dyeing liquid B is an aqueous solution containing 0.3 to 3 weight percent of sodium lauroyl sarcosinate, 1 to 10 weight percent of dithio-DL-threitol, 2 to 10 weight percent of trihydroxymethyl aminomethane, 0.2 to 2 weight percent of disodium ethylene diamine tetraacetate dihydrate and 0.2 to 2 weight percent of Tween 20, and the pH value of the aqueous solution is 7.0 to 8.0;
the sample diluent is 0.5-2 wt% of low-melting point agarose aqueous solution.
2. The sperm DNA fragment staining kit of claim 1, further comprising a preservative ProClin 300 in the phosphate buffer, wherein the preservative ProClin 300 is present in an effective amount of 2 to 5wt%.
3. The sperm DNA fragment staining kit of claim 1, wherein the coated slide is a slide coated with 0.5 to 2wt% aqueous agarose.
4. Use of a sperm DNA fragmentation staining kit according to any of claims 1 to 3, comprising use in the detection of the extent of DNA fragmentation in sperm cells for non-diagnostic purposes and/or use in sperm quality assessment.
5. A sperm DNA fragment staining method for non-diagnostic purposes, wherein staining is performed using the sperm DNA fragment staining kit according to any one of claims 1 to 3, comprising the steps of:
step 1: incubating the sample dilution until it is completely dissolved, and then transferring it to 37 ℃ for equilibration for at least 5 minutes;
step 2: regulating sperm concentration to 5-10 × 10 with physiological saline 6 Per mL, adding a certain amount of the diluted sample solution obtained in the step 1 into the diluted sample solution, fully and uniformly mixing, and incubating at 37 ℃ for later use;
and step 3: adding a certain amount of the sperm diluent obtained in the step (2) on a pre-refrigerated coating glass slide, immediately covering a cover glass, and refrigerating to completely solidify the sperm diluent;
and 4, step 4: removing the cover glass in a side-drawing mode, wherein the cover glass always clings to the glass slide to slide in the removing process, and the cover glass cannot be lifted upwards;
and 5: dripping the staining solution A at room temperature for reaction for 7 minutes, discarding the staining solution A, dripping the staining solution B for reaction for 15 minutes, and discarding the staining solution B;
step 6: horizontally placing the glass slide in purified water for 5 minutes, and changing the water for 1-2 times;
and 7: placing the glass slides in 70% ethanol, 90% ethanol and absolute ethanol in sequence respectively, dehydrating for 2 minutes respectively, taking out, and naturally drying in air;
and 8: mixing the Ruhry-Giemsa staining solution and phosphate buffer solution in proportion according to the number of the slides to be stained to obtain mixed staining solution, dripping the mixed staining solution on each slide to completely cover the slide, after staining for 15 minutes, washing the dye from one side of the slide by using purified water, naturally drying or blow-drying in a fume hood, and observing the staining result of sperms by using a microscope.
6. A method of staining sperm DNA fragments not of diagnostic interest as described in claim 5, wherein said incubating in step 1 is at a temperature of 80 ℃ for a period of 20 minutes.
7. A method of staining sperm DNA fragments not diagnostic of interest according to claim 5, wherein the amount of staining solution A and staining solution B added dropwise in step 5 is 1 to 2mL.
8. A method of staining sperm DNA fragments not of diagnostic interest as defined in claim 5, wherein said Richter Giemsa stain is mixed with phosphate buffer in a ratio of 1 to 2 in step 8, and wherein the drop size of said mixed stain is 1 to 1.5mL.
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