CN104073555A - Sperm DNA fragmentation detection kit - Google Patents
Sperm DNA fragmentation detection kit Download PDFInfo
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- CN104073555A CN104073555A CN201410203476.5A CN201410203476A CN104073555A CN 104073555 A CN104073555 A CN 104073555A CN 201410203476 A CN201410203476 A CN 201410203476A CN 104073555 A CN104073555 A CN 104073555A
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- 238000001514 detection method Methods 0.000 title claims abstract description 45
- 238000013467 fragmentation Methods 0.000 title abstract description 9
- 238000006062 fragmentation reaction Methods 0.000 title abstract description 9
- 239000011521 glass Substances 0.000 claims abstract description 97
- 239000000243 solution Substances 0.000 claims abstract description 29
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 128
- 239000007788 liquid Substances 0.000 claims description 106
- 238000003756 stirring Methods 0.000 claims description 84
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 78
- 239000008213 purified water Substances 0.000 claims description 75
- 238000002360 preparation method Methods 0.000 claims description 42
- 229920000936 Agarose Polymers 0.000 claims description 40
- 239000007864 aqueous solution Substances 0.000 claims description 40
- 239000012634 fragment Substances 0.000 claims description 32
- 238000002156 mixing Methods 0.000 claims description 32
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 28
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 28
- 239000000049 pigment Substances 0.000 claims description 28
- 238000013016 damping Methods 0.000 claims description 24
- 239000012530 fluid Substances 0.000 claims description 24
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 22
- 238000004321 preservation Methods 0.000 claims description 22
- 239000000975 dye Substances 0.000 claims description 20
- 238000006243 chemical reaction Methods 0.000 claims description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims description 14
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 14
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 14
- 229960000583 acetic acid Drugs 0.000 claims description 14
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims description 14
- 239000012362 glacial acetic acid Substances 0.000 claims description 14
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 12
- 229910019142 PO4 Inorganic materials 0.000 claims description 12
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 12
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 12
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 12
- 239000010452 phosphate Substances 0.000 claims description 12
- 229910052700 potassium Inorganic materials 0.000 claims description 12
- 239000011591 potassium Substances 0.000 claims description 12
- 210000000582 semen Anatomy 0.000 claims description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 10
- 239000007983 Tris buffer Substances 0.000 claims description 9
- 239000000872 buffer Substances 0.000 claims description 8
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 8
- 239000002953 phosphate buffered saline Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- 239000002253 acid Substances 0.000 claims description 6
- 239000003513 alkali Substances 0.000 claims description 6
- 235000011194 food seasoning agent Nutrition 0.000 claims description 6
- 239000002504 physiological saline solution Substances 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 238000002844 melting Methods 0.000 claims description 4
- 230000008018 melting Effects 0.000 claims description 4
- 101710171243 Peroxidase 10 Proteins 0.000 claims description 3
- 238000005138 cryopreservation Methods 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 239000001257 hydrogen Substances 0.000 claims description 3
- 229910052739 hydrogen Inorganic materials 0.000 claims description 3
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims description 3
- 101100476979 Rhodobacter capsulatus sdsA gene Proteins 0.000 claims description 2
- 239000000463 material Substances 0.000 abstract description 4
- 108010077544 Chromatin Proteins 0.000 abstract description 2
- 210000003483 chromatin Anatomy 0.000 abstract description 2
- 238000009792 diffusion process Methods 0.000 abstract description 2
- AXIKDPDWFVPGOD-UHFFFAOYSA-O [7-(dimethylamino)phenothiazin-3-ylidene]-dimethylazanium;2-(2,4,5,7-tetrabromo-3,6-dihydroxyxanthen-10-ium-9-yl)benzoic acid Chemical compound C1=CC(=[N+](C)C)C=C2SC3=CC(N(C)C)=CC=C3N=C21.OC(=O)C1=CC=CC=C1C1=C(C=C(Br)C(O)=C2Br)C2=[O+]C2=C1C=C(Br)C(O)=C2Br AXIKDPDWFVPGOD-UHFFFAOYSA-O 0.000 abstract 1
- 239000007853 buffer solution Substances 0.000 abstract 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 10
- 239000004141 Sodium laurylsulphate Substances 0.000 description 10
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 10
- 229910000397 disodium phosphate Inorganic materials 0.000 description 3
- 235000019800 disodium phosphate Nutrition 0.000 description 3
- 238000010586 diagram Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 108010061100 Nucleoproteins Proteins 0.000 description 1
- 102000011931 Nucleoproteins Human genes 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
-
- 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
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
-
- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention provides a sperm DNA fragmentation detection kit. The kit comprises an enveloped glass slide, a fusible gel, an A solution, a B solution, a Wright stain, a Wright buffer solution and a SCD (sperm chromatin diffusion) storage solution. The invention further provides a method for detecting sperm DNA fragmentation by using above kit. The reagent structure is optimized according to the detection method so that the reagent is stable and reliable; the operation of detecting by using the reagent is simple and free from special detection instruments, the clinical conventional application and popularization are facilitated; by adopting the unique SCD storage solution, the sperm sample can be effectively stored, the DNA fragmentation rate cannot be changed after being cryopreserved at -20 DEG.C within two weeks, the required sample can be easily stored through the SCD storage solution, the detection can be performed without the limitation of time and number, and large number of manpower and material resources are saved.
Description
Technical field
The invention belongs to vitro detection reagent class, particularly DNA fragment with spermatozoon detection kit.
Background technology
The fragment degree of sperm DNA has reflected the integrity of sperm genetic substance.Sperm Chromatin diffusion process is one of main method detecting DNA fragment with spermatozoon.The complete sperm of DNA diffuses to form distinctive halation at DNA through sex change and after removing nucleoprotein, and exists the sperm of DNA fragment can not produce this distinctive halation, according to having or not and the DNA integrated degree of size judgement sperm of halation.
At present, there is on the market businessman to release business-like DNA fragment with spermatozoon detection kit, but mostly had some problems, as: background color is too dark, sperm tail is unintelligible, the too little problems such as interpretation difficulty that cause of halation.
Summary of the invention
Main purpose of the present invention is to provide that a kind of methodology is special, result is easy to judge, simple to operate, the DNA fragment with spermatozoon detection kit that is easy to clinical application.
The invention provides a kind of DNA fragment with spermatozoon detection kit, comprising:
Coated slide glass,
Meltable gel,
A liquid,
B liquid,
Wright's stain,
Rui Shi damping fluid,
SCD preserves liquid;
Described coated slide glass is to be coated with the slide glass of the aqueous solution that 0.1～5% fusing point is the agarose of 20～60 ℃; Described meltable gel is the aqueous solution of the agarose of 20～60 ℃ for containing 0.1～5% fusing point; Described A liquid is the aqueous solution containing 0.01%～0.5% sodium-chlor, 0.1%～5% Padil, 0.1%～1% Glacial acetic acid, 0.01%～0.5% hydrogen peroxide; Described B liquid is for containing the aqueous solution that 0.1%～1%SDS, 1%～10%Tris, 0.1%～1% 2 water disodium ethylene diamine tetraacetate, 0.1%～1% polysorbas20 and pH value are 7.0～8.0; Described Wright's stain is the methanol solution containing 0.05～0.5% Rui Shi pigment, 0.01～0.1% Ji Shi pigment; 0.01～0.1mol/L phosphate buffered saline buffer that described Rui Shi damping fluid is pH value 6.0～7.0; Described SCD preserves liquid for the aqueous solution containing 0.1～1% sodium-chlor, 40～80% polysorbas20s.
The preparation method of described coated slide glass is:
1) take low melting-point agarose 1～50g and be placed in the container that can load 1000ml, add 50～200ml purified water, 50 ± 5 ℃ of stirring in water bath are dissolved agarose completely;
2) add purified water constant volume to 1000ml, stirring and evenly mixing, 50 ± 5 ℃ of water bath heat preservations;
3) while hot by step 2) solution is placed in slide surface and paves, dry.
The preparation method of described meltable gel is:
1) take agarose 1～50g and put beaker, be placed in the container that can load 1000ml, add 50～200ml purified water, in water bath, 50 ± 5 ℃ of stirring in water bath are dissolved agarose completely;
2) add purified water constant volume to 1000ml, stirring and evenly mixing, 50 ± 5 ℃ of water bath heat preservations 2 hours;
3) while hot by step 2) solution is sub-packed in 0.5ml sample hose, every pipe 0.14ml.
The preparation method of described A liquid is:
1) get 50～200ml purified water and be placed in 1000ml volumetric flask, take sodium-chlor 0.1～5g, Padil 1～50g, add in above-mentioned volumetric flask, stir it is dissolved completely;
2) measure Glacial acetic acid 1～10ml, 30% hydrogen peroxidase 10 .33～16.7ml, add in above-mentioned volumetric flask, stir it is dissolved completely;
3) with purified water, be settled to 1000ml, stirring and evenly mixing.
The preparation method of described B liquid is:
1) get 50～200ml purified water and be placed in 1000ml volumetric flask, take SDS1～10g, Tris10～100g, two water disodium ethylene diamine tetraacetate 1～10g, add in above-mentioned volumetric flask, stir it is dissolved completely.
2) measure polysorbas20 1～10ml, add in above-mentioned volumetric flask, stir it is dissolved completely.
3) under pH detects, slowly add concentrated hydrochloric acid, regulate pH to 7.0～8.0.
4) with purified water, be settled to 1000ml.
The preparation method of described Wright's stain is:
1) take 0.25～2.5g Rui Shi pigment, 0.05～0.5g Ji Shi pigment, put in clean beaker, add 5～20ml methyl alcohol, grind a moment, sucking-off upper strata dye liquor; Add again 5～20ml methyl alcohol, continue to grind, then liquid in sucking-off, so continuously several times, until dye powder, all dissolve;
2) by methanol constant volume, arrive 500ml, stirring and evenly mixing, is transferred to brown reagent bottle and preserves.
The preparation method of described Rui Shi damping fluid is:
1) get 50～200ml purified water and be placed in 1000ml volumetric flask, take disodium hydrogen phosphate 1.071～10.712g, potassium primary phosphate 1.107～11.067g, add in above-mentioned volumetric flask, stir it is dissolved completely;
2) with pH meter, detect pH, 6.0～7.0 is qualified, dense disodium hydrogen phosphate adjustment for meta-acid, meta-alkali potassium primary phosphate adjustment;
3) with purified water, be settled to 1000ml.
The preparation method that described SCD preserves liquid is:
1) get 50～200ml purified water and be placed in 1000ml volumetric flask, take sodium-chlor 1～10g, add in above-mentioned volumetric flask, stir it is dissolved completely;
2) measure polysorbas20 400～800ml, add in above-mentioned volumetric flask, stir it is dissolved completely;
3) with purified water, be settled to 1000ml, stirring and evenly mixing.
Described DNA fragment with spermatozoon detection kit, preferably includes:
Coated slide glass,
Meltable gel,
A liquid,
B liquid,
Wright's stain,
Rui Shi damping fluid,
SCD preserves liquid;
Described coated slide glass is the slide glass that is coated with the aqueous solution of 0.5% low melting-point agarose; Described meltable gel is the aqueous solution containing 0.25% agarose; Described A liquid is the aqueous solution containing 0.05% sodium-chlor, 0.55% Padil, 0.25% Glacial acetic acid and 0.045% hydrogen peroxide; Described B liquid is for containing the aqueous solution that 0.25%SDS, 2.422%Tris, 0.125% 2 water disodium ethylene diamine tetraacetate, 0.15% polysorbas20 and pH value are 7.2～7.4; Described Wright's stain is the methanol solution containing 0.1% Rui Shi pigment, 0.03% Ji Shi pigment; The 0.03mol/L phosphate buffered saline buffer that described Rui Shi damping fluid is pH value 6.2～6.4; Described SCD preserves liquid for the aqueous solution containing 0.36% sodium-chlor, 45% polysorbas20.
The detection method of described DNA fragment with spermatozoon detection kit, comprises the steps:
1. reagent is prepared
1) sample hose that the meltable gel of 0.14ml is housed is placed in to 80 ℃ and hatches 20 minutes, after melting completely, by meltable gel tube be placed in 37 ℃ stand-by, at least balance is 5 minutes;
2) before detection, room temperature is adjusted to 20～28 ℃ of left and right;
2. sample is prepared
1) with the fresh spermatozoa of physiological saline adjustment liquefaction or the sperm of liquid nitrogen cryopreservation or motile sperm concentration to 5～10 * 10 after extracting
6/ ml;
2) can not complete the fresh specimens of detection, freezing preservation after use SCD preservation liquid, method is as follows: in Eppendorf pipe, add SCD to preserve liquid 300 μ l and seminal fluid 100 μ l to be preserved, fully mix, be placed in-20 ℃ or-80 ℃ of preservations, during detection, by after its equilibrium at room temperature, with physiological saline, adjust sperm concentration to 5～10 * 10
6/ ml;
3. detecting step
1) get off-the-shelf sample to be measured 60 μ l, add in the sample hose that above-mentioned meltable gel melted, fully mix, obtain suspension, hatch stand-by for 37 ℃;
2) will be coated with slide glass and be placed in 2～8 ℃ of refrigerator precoolings and take out after 5 minutes, in the coated region of slide glass, add step 1 rapidly) the suspension 30 μ l that prepare;
3) cover rapidly cover plate, avoid producing bubble; Put 2～8 ℃ of refrigerators 5 minutes, it is solidified;
4) from refrigerator, take out slide glass, carefully remove and cover superincumbent cover plate;
5) slide glass is dipped vertically in the reaction tank that fills reaction solution A immediately, 20～28 ℃ are reacted 7 minutes;
6) take out slide glass, with filter paper, suck the liquid that remains in the slide glass back side and lateral margin; Slide glass is dipped vertically in the reaction tank that fills reaction solution B to 20～28 ℃ of accurate responses 25 minutes;
7) take out slide glass, with filter paper, suck the liquid that remains in the slide glass back side and lateral margin; Slide glass level is immersed in a large amount of purified water 5 minutes, change water therebetween 1～2 time;
8) take out slide glass, with filter paper, suck the liquid that remains in the slide glass back side and lateral margin; Slide glass is dipped vertically in the reaction tank that fills 70% ethanol to 2 minutes;
9) take out slide glass, with filter paper, suck the liquid that remains in the slide glass back side and lateral margin; Slide glass is dipped vertically in the reaction tank that fills 90% ethanol to 2 minutes;
10) take out slide glass, with filter paper, suck the liquid that remains in the slide glass back side and lateral margin; Slide glass is dipped vertically in the reaction tank that fills 100% ethanol to 2 minutes;
11) seasoning in air;
12) every slide glass is with 15～20 coverings of Wright's stain, adds lentamente 30～40 of Rui Shi damping fluids after waiting 1min again, with rubber suction bulb, blows and beats gently mixing dye liquor, after the standing 15min of room temperature, with flowing water, rinses gently and dyes sheet;
13) seasoning or dry up;
14) 500 sperms of 40 * micro-Microscopic observation, there is the sperm quantity of DNA fragment in counting.
Due to technique scheme, the present invention has following technique effect:
1, the present invention has optimized agent structure according to detection method, makes stable reagent, reliable, and simple to operate and without special detection instrument when applying these reagent and detecting, and is convenient to routine clinical application and popularization.
2, the present invention has adopted exclusive SCD to preserve liquid, can effectively preserve semen sample, and its DNA fragmentation rate does not change within-20 ℃ of frozen fortnights.By SCD, preserve liquid like this and can easily preserve required sample, what be not subject to that T/A limits detects, and has saved a large amount of manpower and materials.
Accompanying drawing explanation
Fig. 1 is the concrete steps 4 that DNA fragment with spermatozoon detection kit detects) in along cover plate lower end, promote gently forward the schematic diagram of cover plate.
Fig. 2 is the concrete steps 4 that DNA fragment with spermatozoon detection kit detects) in pinch the overhang of cover plate and along slide glass plane, take lightly the schematic diagram of cover plate away.
Embodiment
The invention provides a kind of DNA fragment with spermatozoon detection kit, comprising:
Coated slide glass;
Meltable gel;
A liquid;
B liquid;
Wright's stain;
Rui Shi damping fluid;
SCD preserves liquid.
Described coated slide glass is for being coated with the slide glass of the aqueous solution of 0.1～5% low melting point (fusing point is 20～60 ℃) agarose; Described meltable gel (fusing point is 20～60 ℃) is the aqueous solution containing 0.1～5% agarose; Described A liquid is the aqueous solution containing 0.01%～0.5% sodium-chlor, 0.1%～5% Padil, 0.1%～1% Glacial acetic acid, 0.01%～0.5% hydrogen peroxide; Described B liquid is for containing the aqueous solution that 0.1%～1%SDS, 1%～10%Tris, 0.1%～1% 2 water disodium ethylene diamine tetraacetate, 0.1%～1% polysorbas20 and pH value are 7.0～8.0; Described Wright's stain is the methanol solution containing 0.05～0.5% Rui Shi pigment, 0.01～0.1% Ji Shi pigment; 0.01～0.1mol/L phosphate buffered saline buffer that described Rui Shi damping fluid is pH value 6.0～7.0; Described SCD preserves liquid for the aqueous solution containing 0.1～1% sodium-chlor, 40～80% polysorbas20s.Existing DNA fragment detection must be used the semen sample of fresh semen sample or Liquid nitrogen, when using fresh semen sample, and inconvenient, be also restricted detection time, and consumption manpower and materials are also many; And when adopting Liquid nitrogen, the cost of preservation is too high.And the SCD that the present invention uses preservation liquid can be preserved semen sample, its DNA fragmentation rate does not change within-20 ℃ of frozen fortnights.By SCD, preserve liquid like this and can easily preserve required sample, what be not subject to that T/A limits detects, for example, can make a collection of specimens, and within several days, detects once, has saved a large amount of manpower and materials.
The preparation method of described coated slide glass is:
1) take low melting-point agarose 1～50g and be placed in the container that can load 1000ml, add 50～200ml purified water, 50 ± 5 ℃ of stirring in water bath are dissolved agarose completely.
2) add purified water constant volume to 1000ml, stirring and evenly mixing, 50 ± 5 ℃ of water bath heat preservations.
3) while hot by step 2) solution is placed in slide surface and paves, dry.
The preparation method of described meltable gel is:
1) take agarose 1～50g and put beaker, be placed in the container that can load 1000ml, add 50～200ml purified water, in water bath, 50 ± 5 ℃ of stirring in water bath are dissolved agarose completely.
2) add purified water constant volume to 1000ml, stirring and evenly mixing, 50 ± 5 ℃ of water bath heat preservations 2 hours.
3) while hot by step 2) solution is sub-packed in 0.5ml sample hose, every pipe 0.14ml.
The preparation method of described A liquid is:
1) get 50～200ml purified water and be placed in 1000ml volumetric flask, take sodium-chlor 0.1～5g, Padil 1～50g, add in above-mentioned volumetric flask, stir it is dissolved completely.
2) measure Glacial acetic acid 1～10ml, 30% hydrogen peroxidase 10 .33～16.7ml, add in above-mentioned volumetric flask, stir it is dissolved completely.
3) with purified water, be settled to 1000ml, stirring and evenly mixing.
The preparation method of described B liquid is:
1) get 50～200ml purified water and be placed in 1000ml volumetric flask, take SDS (sodium lauryl sulphate) 1～10g, Tris (Tutofusin tris) 10～100g, two water disodium ethylene diamine tetraacetate (producers: 1Guanghua Chemical Plant Co., Ltd., Guangdong, model: 250g/ bottle) 1～10g, add in above-mentioned volumetric flask, stir it dissolved completely.
2) measure polysorbas20 1～10ml, add in above-mentioned volumetric flask, stir it is dissolved completely.
3) under pH detects, slowly add concentrated hydrochloric acid, regulate pH to 7.0～8.0.
4) with purified water, be settled to 1000ml.
The preparation method of described Wright's stain is:
1) take 0.25～2.5g Rui Shi pigment, 0.05～0.5g Ji Shi pigment, put in clean beaker, add 5～20ml methyl alcohol, grind a moment, sucking-off upper strata dye liquor; Add again 5～20ml methyl alcohol, continue to grind, then liquid in sucking-off, so continuously several times, until dye powder, all dissolve;
2) by methanol constant volume, arrive 500ml, stirring and evenly mixing, is transferred to brown reagent bottle and preserves.
The preparation method of described Rui Shi damping fluid is:
1) get 50～200ml purified water and be placed in 1000ml volumetric flask, take disodium hydrogen phosphate 1.071～10.712g, potassium primary phosphate 1.107～11.067g, add in above-mentioned volumetric flask, stir it is dissolved completely.
2) with pH meter, detect pH, 6.0～7.0 is qualified, dense disodium hydrogen phosphate adjustment for meta-acid, meta-alkali potassium primary phosphate adjustment.
3) with purified water, be settled to 1000ml.
The preparation method that described SCD preserves liquid is:
1) get 50～200ml purified water and be placed in 1000ml volumetric flask, take sodium-chlor 1～10g, add in above-mentioned volumetric flask, stir it is dissolved completely.
2) measure polysorbas20 400～800ml, add in above-mentioned volumetric flask, stir it is dissolved completely.
3) with purified water, be settled to 1000ml, stirring and evenly mixing.
Below by specific embodiment, the present invention is described in further detail.
Embodiment 1
The present embodiment provides a kind of DNA fragment with spermatozoon detection kit, comprising:
Coated slide glass;
Meltable gel;
A liquid;
B liquid;
Wright's stain;
Rui Shi damping fluid;
SCD preserves liquid.
Described coated slide glass is the slide glass that is coated with the aqueous solution of 0.5% low melting-point agarose; Described meltable gel is the aqueous solution containing 0.25% agarose; Described A liquid is the aqueous solution containing 0.05% sodium-chlor, 0.55% Padil, 0.25% Glacial acetic acid and 0.045% hydrogen peroxide; Described B liquid is for containing the aqueous solution that 0.25%SDS, 2.422%Tris, 0.125% 2 water disodium ethylene diamine tetraacetate, 0.15% polysorbas20 and pH value are 7.2～7.4; Described Wright's stain is the methanol solution containing 0.1% Rui Shi pigment, 0.03% Ji Shi pigment; The 0.03mol/L phosphate buffered saline buffer that described Rui Shi damping fluid is pH value 6.2～6.4; Described SCD preserves liquid for the aqueous solution containing 0.36% sodium-chlor, 45% polysorbas20.Described SCD preserves liquid can preserve semen sample, and in-20 ℃ of frozen fortnights, its DNA fragmentation rate can not change.
The preparation method of described coated slide glass is:
1) take low melting-point agarose [producer: Sigma's aldrich (Shanghai) trade Co., Ltd, model: 25g/ bottle] 5g and be placed in the container that can load 1000ml, add purified water 100ml, 50 ± 5 ℃ of stirring in water bath are dissolved agarose completely.
2) add purified water constant volume to 1000ml, stirring and evenly mixing, 50 ± 5 ℃ of water bath heat preservations.
3) while hot by step 2) solution is placed in slide glass surface and paves, dry.
The preparation method of described meltable gel is:
1) take agarose [producer: Sigma's aldrich (Shanghai) trade Co., Ltd, model: 25g/ bottle] 2.5g puts beaker, be placed in the container that can load 1000ml, add purified water 100ml, in water bath, 50 ± 5 ℃ of stirring in water bath are dissolved agarose completely.
2) add purified water constant volume to 1000ml, stirring and evenly mixing, 50 ± 5 ℃ of water bath heat preservations 2 hours.
3) while hot by step 5) solution is sub-packed in 0.5ml sample hose, every pipe 0.14ml.
The preparation method of described A liquid is:
1) get 100ml purified water and be placed in 1000ml volumetric flask, take sodium-chlor 0.5g, Padil 5.5g, add in above-mentioned volumetric flask, stir it is dissolved completely.
2) measure Glacial acetic acid 2.5ml, 30% hydrogen peroxide 1.5ml, add in above-mentioned volumetric flask, stir it is dissolved completely.
3) with purified water, be settled to 1000ml, stirring and evenly mixing.
The preparation method of described B liquid is:
1) get 100ml purified water and be placed in 1000ml volumetric flask, take SDS2.5g, Tris24.22g, two water disodium ethylene diamine tetraacetate 1.25g, add in above-mentioned volumetric flask, stir it is dissolved completely.
2) measure polysorbas20 1.5ml, add in above-mentioned volumetric flask, stir it is dissolved completely.
3) under pH detects, slowly add concentrated hydrochloric acid, regulate pH to 7.2～7.4.
4) with purified water, be settled to 1000ml.
The preparation method of described Wright's stain is:
1) take 0.5g Rui Shi pigment, 0.15g Ji Shi pigment, put in clean beaker, add 10ml methyl alcohol, grind a moment, sucking-off upper strata dye liquor; Add again 10ml methyl alcohol, continue to grind, then liquid in sucking-off, so continuously several times, until dye powder, all dissolve;
2) by methanol constant volume, arrive 500ml, stirring and evenly mixing, is transferred to brown reagent bottle and preserves.
The preparation method of described Rui Shi damping fluid is:
1) get appropriate purified water and be placed in 1000ml volumetric flask, take disodium hydrogen phosphate 3.213g, potassium primary phosphate 3.320g, add in above-mentioned volumetric flask, stir it is dissolved completely.
2) with pH meter, detect pH, 6.2～6.4 is qualified, dense 12 water Sodium phosphate dibasic adjustment for meta-acid, meta-alkali potassium primary phosphate adjustment.
3) with purified water, be settled to 1000ml.
The preparation method that described SCD preserves liquid is:
1) get 100ml purified water and be placed in 1000ml volumetric flask, take sodium-chlor 3.6g, add in above-mentioned volumetric flask, stir it is dissolved completely.
2) measure polysorbas20 450ml, add in above-mentioned volumetric flask, stir it is dissolved completely.
3) with purified water, be settled to 1000ml, stirring and evenly mixing.
Embodiment 2
The present embodiment provides a kind of DNA fragment with spermatozoon detection kit, comprising:
Coated slide glass;
Meltable gel;
A liquid;
B liquid;
Wright's stain;
Rui Shi damping fluid;
SCD preserves liquid.
Described coated slide glass is the slide glass that is coated with the aqueous solution of 2% low melting-point agarose; Described meltable gel is the aqueous solution containing 1% agarose; Described A liquid is the aqueous solution containing 0.2% sodium-chlor, 2.2% Padil, 0.10% Glacial acetic acid and 0.18% hydrogen peroxide; Described B liquid is for containing the aqueous solution that 1% sodium lauryl sulphate (SDS), 10%Tris, 0.5% 2 water disodium ethylene diamine tetraacetate, 0.6% polysorbas20 and pH value are 7.6～8.0; Described Wright's stain is the methanol solution containing 0.4% Rui Shi pigment, 0.12% Ji Shi pigment; The 0.12mol/L phosphate buffered saline buffer that described Rui Shi damping fluid is pH value 6.8～7.0; It is the aqueous solution of 1.44% sodium-chlor, 75% polysorbas20 that described SCD preserves liquid.Described SCD preserves liquid can preserve semen sample, and in-20 ℃ of frozen fortnights, its DNA fragmentation rate can not change.
The preparation method of described coated slide glass is:
1) take low melting-point agarose [producer: Sigma's aldrich (Shanghai) trade Co., Ltd, model: 25g/ bottle] 20g and be placed in the container that can load 1000ml, add purified water 100ml, 50 ± 5 ℃ of stirring in water bath are dissolved agarose completely.
2) add purified water constant volume to 1000ml, stirring and evenly mixing, 50 ± 5 ℃ of water bath heat preservations.
3) while hot by step 2) solution is placed in slide glass surface and paves, dry.
The preparation method of described meltable gel is:
1) take agarose [producer: Sigma's aldrich (Shanghai) trade Co., Ltd, model: 25g/ bottle] 10g puts beaker, be placed in the container that can load 1000ml, add purified water 100ml, in water bath, 50 ± 5 ℃ of stirring in water bath are dissolved agarose completely.
2) add purified water constant volume to 1000ml, stirring and evenly mixing, 50 ± 5 ℃ of water bath heat preservations 2 hours.
3) while hot by step 5) solution is sub-packed in 0.5ml sample hose, every pipe 0.14ml.
The preparation method of described A liquid is:
1) get 100ml purified water and be placed in 1000ml volumetric flask, take sodium-chlor 2g, Padil 22g, add in above-mentioned volumetric flask, stir it is dissolved completely.
2) measure Glacial acetic acid 1.0ml, 30% hydrogen peroxide 6.0ml, add in above-mentioned volumetric flask, stir it is dissolved completely.
3) with purified water, be settled to 1000ml, stirring and evenly mixing.
The preparation method of described B liquid is:
1) get 100ml purified water and be placed in 1000ml volumetric flask, take SDS10g, Tris100g, 2 water disodium ethylene diamine tetraacetate 5g, add in above-mentioned volumetric flask, stir it is dissolved completely.
2) measure polysorbas20 6.0ml, add in above-mentioned volumetric flask, stir it is dissolved completely.
3) under pH detects, slowly add concentrated hydrochloric acid, regulate pH to 7.6～8.0.
4) with purified water, be settled to 1000ml.
The preparation method of described Wright's stain is:
1) take 2.0g Rui Shi pigment, 0.6g Ji Shi pigment, put in clean beaker, add 10ml methyl alcohol, grind a moment, sucking-off upper strata dye liquor; Add again 10ml methyl alcohol, continue to grind, then liquid in sucking-off, so continuously several times, until dye powder, all dissolve;
2) by methanol constant volume, arrive 500ml, stirring and evenly mixing, is transferred to brown reagent bottle and preserves.
The preparation method of described Rui Shi damping fluid is:
1) get appropriate purified water and be placed in 1000ml volumetric flask, take disodium hydrogen phosphate 12.854g, potassium primary phosphate 13.28g, add in above-mentioned volumetric flask, stir it is dissolved completely.
2) with pH meter, detect pH, 6.8～7.0 is qualified, dense 12 water Sodium phosphate dibasic adjustment for meta-acid, meta-alkali potassium primary phosphate adjustment.
3) with purified water, be settled to 1000ml.
The preparation method that described SCD preserves liquid is:
1) get 100ml purified water and be placed in 1000ml volumetric flask, take sodium-chlor 14.4g, add in above-mentioned volumetric flask, stir it is dissolved completely.
2) measure polysorbas20 750ml, add in above-mentioned volumetric flask, stir it is dissolved completely.
3) with purified water, be settled to 1000ml, stirring and evenly mixing.
Embodiment 3
The present embodiment provides a kind of DNA fragment with spermatozoon detection kit, comprising:
Coated slide glass;
Meltable gel;
A liquid;
B liquid;
Wright's stain;
Rui Shi damping fluid;
SCD preserves liquid.
Described coated slide glass is the slide glass that is coated with the aqueous solution of 1% low melting-point agarose; Described meltable gel is the aqueous solution containing 0.5% agarose; Described A liquid is the aqueous solution containing 0.1% sodium-chlor, 1.1% Padil, 0.50% Glacial acetic acid and 0.09% hydrogen peroxide; Described B liquid is for containing the aqueous solution that 0.5% sodium lauryl sulphate (SDS), 4.844%Tris, 0.2525% 2 water disodium ethylene diamine tetraacetate, 0.3% polysorbas20 and pH value are 7.3～7.7; Described Wright's stain is the methanol solution containing 0.2% Rui Shi pigment, 0.06% Ji Shi pigment; The 0.06mol/L phosphate buffered saline buffer that described Rui Shi damping fluid is pH value 6.4～6.8; It is the aqueous solution of 0.72% sodium-chlor, 65% polysorbas20 that described SCD preserves liquid.Described SCD preserves liquid can preserve semen sample, and in-20 ℃ of frozen fortnights, its DNA fragmentation rate can not change.
The preparation method of described coated slide glass is:
1) take low melting-point agarose [producer: Sigma's aldrich (Shanghai) trade Co., Ltd, model: 25g/ bottle] 10g and be placed in the container that can load 1000ml, add purified water 100ml, 50 ± 5 ℃ of stirring in water bath are dissolved agarose completely.
2) add purified water constant volume to 1000ml, stirring and evenly mixing, 50 ± 5 ℃ of water bath heat preservations.
3) while hot by step 2) solution is placed in slide glass surface and paves, dry.
The preparation method of described meltable gel is:
1) take agarose [producer: Sigma's aldrich (Shanghai) trade Co., Ltd, model: 25g/ bottle] 5g puts beaker, be placed in the container that can load 1000ml, add purified water 100ml, in water bath, 50 ± 5 ℃ of stirring in water bath are dissolved agarose completely.
2) add purified water constant volume to 1000ml, stirring and evenly mixing, 50 ± 5 ℃ of water bath heat preservations 2 hours.
3) while hot by step 5) solution is sub-packed in 0.5ml sample hose, every pipe 0.14ml.
The preparation method of described A liquid is:
1) get 100ml purified water and be placed in 1000ml volumetric flask, take sodium-chlor 1g, Padil 11g, add in above-mentioned volumetric flask, stir it is dissolved completely.
2) measure Glacial acetic acid 5.0ml, 30% hydrogen peroxide 3.0ml, add in above-mentioned volumetric flask, stir it is dissolved completely.
3) with purified water, be settled to 1000ml, stirring and evenly mixing.
The preparation method of described B liquid is:
1) get 100ml purified water and be placed in 1000ml volumetric flask, take SDS5g, Tris48.44g, 2 water disodium ethylene diamine tetraacetate 2.525g, add in above-mentioned volumetric flask, stir it is dissolved completely.
2) measure polysorbas20 3.0ml, add in above-mentioned volumetric flask, stir it is dissolved completely.
3) under pH detects, slowly add concentrated hydrochloric acid, regulate pH to 7.5 ± 0.2.
4) with purified water, be settled to 1000ml.
The preparation method of described Wright's stain is:
1) take 1.0g Rui Shi pigment, 0.3g Ji Shi pigment, put in clean beaker, add 10ml methyl alcohol, grind a moment, sucking-off upper strata dye liquor; Add again 10ml methyl alcohol, continue to grind, then liquid in sucking-off, so continuously several times, until dye powder, all dissolve;
2) by methanol constant volume, arrive 500ml, stirring and evenly mixing, is transferred to brown reagent bottle and preserves.
The preparation method of described Rui Shi damping fluid is:
1) get appropriate purified water and be placed in 1000ml volumetric flask, take disodium hydrogen phosphate 6.427g, potassium primary phosphate 6.640g, add in above-mentioned volumetric flask, stir it is dissolved completely.
2) with pH meter, detect pH, 6.4～6.8 is qualified, dense 12 water Sodium phosphate dibasic adjustment for meta-acid, meta-alkali potassium primary phosphate adjustment.
3) with purified water, be settled to 1000ml.
The preparation method that described SCD preserves liquid is:
1) get 100ml purified water and be placed in 1000ml volumetric flask, take sodium-chlor 7.2g, add in above-mentioned volumetric flask, stir it is dissolved completely.
2) measure polysorbas20 650ml, add in above-mentioned volumetric flask, stir it is dissolved completely.
3) with purified water, be settled to 1000ml, stirring and evenly mixing.
Embodiment 4
The concrete steps that the present embodiment provides DNA fragment with spermatozoon detection kit to detect are as follows:
1. reagent is prepared
1) sample hose (having the meltable gel of 0.14ml in pipe) that meltable gel is housed is placed in to 80 ℃ and hatches 20 minutes, after melting completely, meltable gel tube is placed in to 37 ℃ stand-by (at least balance is 5 minutes).
2) before detection, room temperature is adjusted to 20～28 ℃ of left and right.
2. sample is prepared
1) fresh spermatozoa liquefying with physiological saline adjustment (or the sperm of liquid nitrogen cryopreservation or the motile sperm after extracting) concentration to 5～10 * 10
6/ ml.
2) can not complete the fresh specimens of detection, can use SCD to preserve freezing preservation after liquid, can at least preserve 15 days.Method is as follows: in Eppendorf pipe, add SCD to preserve liquid 300 μ l and seminal fluid 100 μ l to be preserved, fully mix, be placed in-20 ℃ or-80 ℃ of preservations.During detection, by after its equilibrium at room temperature, with physiological saline, adjust sperm concentration to 5～10 * 10
6/ ml.
3. detecting step
1) get off-the-shelf sample to be measured 60 μ l, add in the sample hose that above-mentioned meltable gel melted and (notice that 37 ℃ continue insulation), fully mix, obtain suspension, hatch stand-by for 37 ℃.
2) will be coated with slide glass (specification is 76.2 * 25.4mm) and be placed in 2～8 ℃ of refrigerator precoolings and take out after 5 minutes, in the coated region of slide glass, add step 1 rapidly) the suspension 30 μ l that prepare.
3) cover rapidly cover plate, avoid producing bubble; Put 2～8 ℃ of refrigerators 5 minutes, it is solidified.
4) from refrigerator, take out slide glass, carefully remove and cover superincumbent cover plate.Method: as shown in Figure 1, promote gently forward cover plate along cover plate lower end, until the cover plate the other end exceeds slide glass width slightly; Pinch the overhang of cover plate, along slide glass plane, take lightly cover plate away, as shown in Figure 2.Attention: in the process of mobile cover plate, cover plate is close to slide glass planar slide all the time, can't upwards be lifted away from gel plane by cover plate.
5) slide glass is dipped vertically into immediately in the reaction tank that fills reaction solution A (A liquid), 20～28 ℃ are reacted 7 minutes.
6) take out slide glass, with filter paper, suck the liquid (not contacting sample district) that remains in the slide glass back side and lateral margin; Slide glass is dipped vertically in the reaction tank that fills reaction solution B (B liquid) to 20～28 ℃ of accurate responses 25 minutes.
7) take out slide glass, with filter paper, suck the liquid (not contacting sample district) that remains in the slide glass back side and lateral margin; Slide glass level is immersed in a large amount of purified water 5 minutes, change water therebetween 1～2 time.
8) take out slide glass, with filter paper, suck the liquid (not contacting sample district) that remains in the slide glass back side and lateral margin; Slide glass is dipped vertically in the reaction tank that fills 70% ethanol to 2 minutes.
9) take out slide glass, with filter paper, suck the liquid (not contacting sample district) that remains in the slide glass back side and lateral margin; Slide glass is dipped vertically in the reaction tank that fills 90% ethanol to 2 minutes.
10) take out slide glass, with filter paper, suck the liquid (not contacting sample district) that remains in the slide glass back side and lateral margin; Slide glass is dipped vertically in the reaction tank that fills 100% ethanol to 2 minutes;
11) seasoning in air.
12) dye sheet.Every slide glass is with 15～20 coverings of Wright's stain, Deng adding lentamente again 30～40 of Rui Shi damping fluids after 1min, with rubber suction bulb, blow and beat gently and mix dye liquor (noting not destroying the surface tension that dye liquor forms), after the standing 15min of room temperature, with flowing water, rinse gently and dye sheet.
13) seasoning or dry up.
14) 500 sperms of 40 * micro-Microscopic observation, there is the sperm quantity of DNA fragment in counting.
4. result is observed and is calculated:
1) DNA fragment with spermatozoon criterion: sperm head only produces less halation or without halation, the thickness of one-sided halation is no more than 1/3 of sperm head minimum diameter.
Claims (10)
1. a DNA fragment with spermatozoon detection kit, is characterized in that, comprising:
Coated slide glass,
Meltable gel,
A liquid,
B liquid,
Wright's stain,
Rui Shi damping fluid,
SCD preserves liquid;
Described coated slide glass is to be coated with the slide glass of the aqueous solution that 0.1～5% fusing point is the agarose of 20～60 ℃; Described meltable gel is the aqueous solution of the agarose of 20～60 ℃ for containing 0.1～5% fusing point; Described A liquid is the aqueous solution containing 0.01%～0.5% sodium-chlor, 0.1%～5% Padil, 0.1%～1% Glacial acetic acid, 0.01%～0.5% hydrogen peroxide; Described B liquid is for containing the aqueous solution that 0.1%～1%SDS, 1%～10%Tris, 0.1%～1% 2 water disodium ethylene diamine tetraacetate, 0.1%～1% polysorbas20 and pH value are 7.0～8.0; Described Wright's stain is the methanol solution containing 0.05～0.5% Rui Shi pigment, 0.01～0.1% Ji Shi pigment; 0.01～0.1mol/L phosphate buffered saline buffer that described Rui Shi damping fluid is pH value 6.0～7.0; Described SCD preserves liquid for the aqueous solution containing 0.1～1% sodium-chlor, 40～80% polysorbas20s.
2. DNA fragment with spermatozoon detection kit according to claim 1, is characterized in that, the preparation method of described coated slide glass is:
1) take low melting-point agarose 1～50g and be placed in the container that can load 1000ml, add 50～200ml purified water, 50 ± 5 ℃ of stirring in water bath are dissolved agarose completely;
2) add purified water constant volume to 1000ml, stirring and evenly mixing, 50 ± 5 ℃ of water bath heat preservations;
3) while hot by step 2) solution is placed in slide surface and paves, dry.
3. DNA fragment with spermatozoon detection kit according to claim 1, is characterized in that, the preparation method of described meltable gel is:
1) take agarose 1～50g and put beaker, be placed in the container that can load 1000ml, add 50～200ml purified water, in water bath, 50 ± 5 ℃ of stirring in water bath are dissolved agarose completely;
2) add purified water constant volume to 1000ml, stirring and evenly mixing, 50 ± 5 ℃ of water bath heat preservations 2 hours;
3) while hot by step 2) solution is sub-packed in 0.5ml sample hose, every pipe 0.14ml.
4. DNA fragment with spermatozoon detection kit according to claim 1, is characterized in that, the preparation method of described A liquid is:
1) get 50～200ml purified water and be placed in 1000ml volumetric flask, take sodium-chlor 0.1～5g, Padil 1～50g, add in above-mentioned volumetric flask, stir it is dissolved completely;
2) measure Glacial acetic acid 1～10ml, 30% hydrogen peroxidase 10 .33～16.7ml, add in above-mentioned volumetric flask, stir it is dissolved completely;
3) with purified water, be settled to 1000ml, stirring and evenly mixing.
5. DNA fragment with spermatozoon detection kit according to claim 1, is characterized in that, the preparation method of described B liquid is:
1) get 50～200ml purified water and be placed in 1000ml volumetric flask, take SDS1～10g, Tris10～100g, two water disodium ethylene diamine tetraacetate 1～10g, add in above-mentioned volumetric flask, stir it is dissolved completely.
2) measure polysorbas20 1～10ml, add in above-mentioned volumetric flask, stir it is dissolved completely.
3) under pH detects, slowly add concentrated hydrochloric acid, regulate pH to 7.0～8.0.
4) with purified water, be settled to 1000ml.
6. DNA fragment with spermatozoon detection kit according to claim 1, is characterized in that, the preparation method of described Wright's stain is:
1) take 0.25～2.5g Rui Shi pigment, 0.05～0.5g Ji Shi pigment, put in clean beaker, add 5～20ml methyl alcohol, grind a moment, sucking-off upper strata dye liquor; Add again 5～20ml methyl alcohol, continue to grind, then liquid in sucking-off, so continuously several times, until dye powder, all dissolve;
2) by methanol constant volume, arrive 500ml, stirring and evenly mixing, is transferred to brown reagent bottle and preserves.
7. DNA fragment with spermatozoon detection kit according to claim 1, is characterized in that, the preparation method of described Rui Shi damping fluid is:
1) get 50～200ml purified water and be placed in 1000ml volumetric flask, take disodium hydrogen phosphate 1.071～10.712g, potassium primary phosphate 1.107～11.067g, add in above-mentioned volumetric flask, stir it is dissolved completely;
2) with pH meter, detect pH, 6.0～7.0 is qualified, dense disodium hydrogen phosphate adjustment for meta-acid, meta-alkali potassium primary phosphate adjustment;
3) with purified water, be settled to 1000ml.
8. DNA fragment with spermatozoon detection kit according to claim 1, is characterized in that, the preparation method that described SCD preserves liquid is:
1) get 50～200ml purified water and be placed in 1000ml volumetric flask, take sodium-chlor 1～10g, add in above-mentioned volumetric flask, stir it is dissolved completely;
2) measure polysorbas20 400～800ml, add in above-mentioned volumetric flask, stir it is dissolved completely;
3) with purified water, be settled to 1000ml, stirring and evenly mixing.
9. DNA fragment with spermatozoon detection kit according to claim 1, is characterized in that, described coated slide glass is the slide glass that is coated with the aqueous solution of 0.5% low melting-point agarose; Described meltable gel is the aqueous solution containing 0.25% agarose; Described A liquid is the aqueous solution containing 0.05% sodium-chlor, 0.55% Padil, 0.25% Glacial acetic acid and 0.045% hydrogen peroxide; Described B liquid is for containing the aqueous solution that 0.25%SDS, 2.422%Tris, 0.125% 2 water disodium ethylene diamine tetraacetate, 0.15% polysorbas20 and pH value are 7.2～7.4; Described Wright's stain is the methanol solution containing 0.1% Rui Shi pigment, 0.03% Ji Shi pigment; The 0.03mol/L phosphate buffered saline buffer that described Rui Shi damping fluid is pH value 6.2～6.4; Described SCD preserves liquid for the aqueous solution containing 0.36% sodium-chlor, 45% polysorbas20.
10. the detection method of DNA fragment with spermatozoon detection kit according to claim 1, comprises the steps:
1. reagent is prepared
1) sample hose that the meltable gel of 0.14ml is housed is placed in to 80 ℃ and hatches 20 minutes, after melting completely, by meltable gel tube be placed in 37 ℃ stand-by, at least balance is 5 minutes;
2) before detection, room temperature is adjusted to 20～28 ℃ of left and right;
2. sample is prepared
1) with the fresh spermatozoa of physiological saline adjustment liquefaction or the sperm of liquid nitrogen cryopreservation or motile sperm concentration to 5～10 * 10 after extracting
6/ ml;
2) can not complete the fresh specimens of detection, freezing preservation after use SCD preservation liquid, method is as follows: in Eppendorf pipe, add SCD to preserve liquid 300 μ l and seminal fluid 100 μ l to be preserved, fully mix, be placed in-20 ℃ or-80 ℃ of preservations, during detection, by after its equilibrium at room temperature, with physiological saline, adjust sperm concentration to 5～10 * 10
6/ ml;
3. detecting step
1) get off-the-shelf sample to be measured 60 μ l, add in the sample hose that above-mentioned meltable gel melted, fully mix, obtain suspension, hatch stand-by for 37 ℃;
2) will be coated with slide glass and be placed in 2～8 ℃ of refrigerator precoolings and take out after 5 minutes, in the coated region of slide glass, add step 1 rapidly) the suspension 30 μ l that prepare;
3) cover rapidly cover plate, avoid producing bubble; Put 2～8 ℃ of refrigerators 5 minutes, it is solidified;
4) from refrigerator, take out slide glass, carefully remove and cover superincumbent cover plate;
5) slide glass is dipped vertically in the reaction tank that fills A liquid immediately, 20～28 ℃ are reacted 7 minutes;
6) take out slide glass, with filter paper, suck the liquid that remains in the slide glass back side and lateral margin, slide glass is dipped vertically in the reaction tank that fills B liquid, 20～28 ℃ of accurate responses 25 minutes;
7) take out slide glass, with filter paper, suck the liquid that remains in the slide glass back side and lateral margin, slide glass level is immersed in a large amount of purified water 5 minutes, change water therebetween 1～2 time;
8) take out slide glass, with filter paper, suck the liquid that remains in the slide glass back side and lateral margin, slide glass is dipped vertically in the reaction tank that fills 70% ethanol to 2 minutes;
9) take out slide glass, with filter paper, suck the liquid that remains in the slide glass back side and lateral margin, slide glass is dipped vertically in the reaction tank that fills 90% ethanol to 2 minutes;
10) take out slide glass, with filter paper, suck the liquid that remains in the slide glass back side and lateral margin, slide glass is dipped vertically in the reaction tank that fills 100% ethanol to 2 minutes;
11) seasoning in air;
12) every slide glass is with 15～20 coverings of Wright's stain, adds lentamente 30～40 of Rui Shi damping fluids after waiting 1min again, with rubber suction bulb, blows and beats gently mixing dye liquor, after the standing 15min of room temperature, with flowing water, rinses gently and dyes sheet;
13) seasoning or dry up;
14) 500 sperms of 40 * micro-Microscopic observation, there is the sperm quantity of DNA fragment in counting.
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| CN104988229A (en) * | 2015-07-10 | 2015-10-21 | 成都朴华科技有限公司 | Sperm DNA fragment detection kit and detecting method thereof |
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| CN109406246A (en) * | 2018-10-25 | 2019-03-01 | 潍坊市康华生物技术有限公司 | A kind of sperm morphological rapid dyeing reagent and its colouring method |
| CN111307696A (en) * | 2020-03-19 | 2020-06-19 | 浙江星博生物科技股份有限公司 | Method and kit for detecting sperm DNA fragmentation rate |
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| CN104388302A (en) * | 2014-12-03 | 2015-03-04 | 张丽红 | Reaction box for sperm chromatin diffusion experiment |
| CN104823966A (en) * | 2015-05-19 | 2015-08-12 | 上海交通大学医学院附属第九人民医院 | Trace sperm cryopreservation carrier and corresponding freezing and thawing methods thereof |
| CN104988229A (en) * | 2015-07-10 | 2015-10-21 | 成都朴华科技有限公司 | Sperm DNA fragment detection kit and detecting method thereof |
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| CN108398407B (en) * | 2018-01-25 | 2020-11-17 | 迈克博（天津）生物科技有限公司 | Human sperm motility rate and DNA damage double-parameter detection and analysis kit |
| CN109406246A (en) * | 2018-10-25 | 2019-03-01 | 潍坊市康华生物技术有限公司 | A kind of sperm morphological rapid dyeing reagent and its colouring method |
| CN111307696A (en) * | 2020-03-19 | 2020-06-19 | 浙江星博生物科技股份有限公司 | Method and kit for detecting sperm DNA fragmentation rate |
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