CN104073555B - DNA fragment with spermatozoon detection kit - Google Patents

DNA fragment with spermatozoon detection kit Download PDF

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CN104073555B
CN104073555B CN201410203476.5A CN201410203476A CN104073555B CN 104073555 B CN104073555 B CN 104073555B CN 201410203476 A CN201410203476 A CN 201410203476A CN 104073555 B CN104073555 B CN 104073555B
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liquid
microscope slide
add
purified water
dna fragment
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CN104073555A (en
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程浩
陶思恩
邓少君
张翔
庄学敏
钟彩颜
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BRED LIFE SCIENCE TECHNOLOGY Inc
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BRED LIFE SCIENCE TECHNOLOGY Inc
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    • 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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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract

The invention provides a kind of DNA fragment with spermatozoon detection kit, including: being coated microscope slide, meltable gel, A liquid, B liquid, Wright's stain, Rui Shi buffer, SCD preserves liquid.Present invention also offers the application mentioned reagent box detection method to DNA fragment with spermatozoon.The present invention optimizes agent structure according to detection method so that stable reagent, reliable, and applies when these reagent detect simple to operate and without special detection instrument, it is simple to routine clinical application and popularization；Present invention employs exclusive SCD and preserve liquid, can effectively preserve semen sample, its DNA fragment rate does not change within-20 DEG C of frozen fortnights, so preserve liquid by SCD and can easily preserve required specimen, the carrying out do not limit by T/A detects, and saves substantial amounts of manpower and materials.

Description

DNA fragment with spermatozoon detection kit
Technical field
The invention belongs to vitro detection reagent class, particularly to DNA fragment with spermatozoon detection kit.
Background technology
The fragment degree of sperm DNA reflects the integrity of sperm genetic substance.Sperm Chromatin diffusion method is one of main method of detection DNA fragment with spermatozoon.Sperm complete for DNA through degeneration and after removing nucleoprotein DNA diffuse to form distinctive halation, and the sperm that there is DNA fragment will not produce this distinctive halation, judges the DNA integrated degree of sperm according to the presence or absence of halation and size.
At present, there is businessman to release business-like DNA fragment with spermatozoon detection kit on the market, but mostly there are some problems, as: background color is too deep, sperm tail is unintelligible, halation is too little causes the problems such as interpretation difficulty.
Summary of the invention
Present invention is primarily targeted at and provide that a kind of methodology is special, result is prone to judge, simple to operate, be prone to the DNA fragment with spermatozoon detection kit of clinical application.
The invention provides a kind of DNA fragment with spermatozoon detection kit, including:
It is coated microscope slide,
Meltable gel,
A liquid,
B liquid,
Wright's stain,
Rui Shi buffer,
SCD preserves liquid；
The described microscope slide being coated the aqueous solution that microscope slide is the agarose that fusing point is 20～60 DEG C being coated with 0.1～5%；Described meltable gel is the aqueous solution of the agarose that fusing point is 20～60 DEG C containing 0.1～5%；Described A liquid be containing 0.01%～0.5% sodium chloride, 0.1%～5% glycine, 0.1%～1% glacial acetic acid, 0.01%～0.5% hydrogen peroxide aqueous solution；Described B liquid is be the aqueous solution of 7.0～8.0 containing 0.1%～1%SDS, 1%～10%Tris, 0.1%～1% 2 water disodiumedetate, 0.1%～1% polysorbas20 and pH value；Described Wright's stain is the methanol solution containing 0.05～0.5% Rui Shi pigment, 0.01～0.1% Ji Shi pigment；Described Rui Shi buffer is 0.01～0.1mol/L phosphate buffer of pH value 6.0～7.0；It is the aqueous solution containing 0.1～1% sodium chloride, 40～80% polysorbas20s that described SCD preserves liquid.
Described be coated microscope slide preparation method be:
1) weighing low melting-point agarose 1～50g and be placed in the container that can load 1000ml, add 50～200ml purified water, 50 ± 5 DEG C of stirring in water bath make agarose be completely dissolved；
2) purified water constant volume is added to 1000ml, stirring and evenly mixing, 50 ± 5 DEG C 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) weighing 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 DEG C of stirring in water bath make agarose be completely dissolved；
2) purified water constant volume is added to 1000ml, stirring and evenly mixing, 50 ± 5 DEG C of water bath heat preservations 2 hours；
3) while hot by step 2) solution is sub-packed in 0.5ml sample cell, often pipe 0.14ml.
The preparation method of described A liquid is:
1) taking 50～200ml purified water to be placed in 1000ml volumetric flask, weigh sodium chloride 0.1～5g, glycine 1～50g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved；
2) measuring glacial acetic acid 1～10ml, 30% hydrogen peroxidase 10 .33～16.7ml, add in above-mentioned volumetric flask, stirring makes it be completely dissolved；
3) it is settled to 1000ml, stirring and evenly mixing with purified water.
The preparation method of described B liquid is:
1) taking 50～200ml purified water to be placed in 1000ml volumetric flask, weigh SDS1～10g, Tris10～100g, two water disodiumedetate 1～10g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
2) measuring polysorbas20 1～10ml, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
3) under pH detects, it is slowly added into concentrated hydrochloric acid, regulates pH to 7.0～8.0.
4) it is settled to 1000ml with purified water.
The preparation method of described Wright's stain is:
1) weigh 0.25～2.5g Rui Shi pigment, 0.05～0.5g Ji Shi pigment, put in clean beaker, add 5～20ml methanol, grind a moment, sucking-off upper strata dye liquor；Add 5 again～20ml methanol, continue to grind, then liquid in sucking-off, so continuously several times, until dye powder all dissolves；
2) with methanol constant volume to 500ml, stirring and evenly mixing, it is transferred to brown reagent bottle and preserves.
The preparation method of described Rui Shi buffer is:
1) taking 50～200ml purified water to be placed in 1000ml volumetric flask, weigh disodium hydrogen phosphate 1.071～10.712g, potassium dihydrogen phosphate 1.107～11.067g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved；
2) detecting solution ph with pH meter, 6.0～7.0 is qualified, meta-acid dense disodium hydrogen phosphate adjustment, meta-alkali potassium dihydrogen phosphate adjustment；
3) it is settled to 1000ml with purified water.
Described SCD preserves the preparation method of liquid:
1) taking 50～200ml purified water to be placed in 1000ml volumetric flask, weigh sodium chloride 1～10g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved；
2) measuring polysorbas20 400～800ml, add in above-mentioned volumetric flask, stirring makes it be completely dissolved；
3) it is settled to 1000ml, stirring and evenly mixing with purified water.
Described DNA fragment with spermatozoon detection kit, it is preferable that including:
It is coated microscope slide,
Meltable gel,
A liquid,
B liquid,
Wright's stain,
Rui Shi buffer,
SCD preserves liquid；
Described it is coated the microscope slide that microscope slide is the aqueous solution being coated with 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 chloride, 0.55% glycine, 0.25% glacial acetic acid and 0.045% hydrogen peroxide；Described B liquid is be the aqueous solution of 7.2～7.4 containing 0.25%SDS, 2.422%Tris, 0.125% 2 water disodiumedetate, 0.15% polysorbas20 and pH value；Described Wright's stain is the methanol solution containing 0.1% Rui Shi pigment, 0.03% Ji Shi pigment；Described Rui Shi buffer is the 0.03mol/L phosphate buffer of pH value 6.2～6.4；It is the aqueous solution containing 0.36% sodium chloride, 45% polysorbas20 that described SCD preserves liquid.
The detection method of described DNA fragment with spermatozoon detection kit, comprises the steps:
1. reagent prepares
1) will be equipped with the sample cell of the meltable gel of 0.14ml to be placed in 80 DEG C and hatch 20 minutes, after melting completely, meltable gel tube is placed in 37 DEG C stand-by, at least balance 5 minutes；
2) before detection, room temperature is adjusted to about 20～28 DEG C；
2. specimen prepares
1) with normal saline adjustment liquefaction fresh spermatozoa or liquid nitrogen cryopreservation sperm or extracted after motile sperm concentration to 5～10 × 106/ml；
2) fresh specimens of detection can not be completed, freezen protective after use SCD preservation liquid, method is as follows: adds SCD in Eppendorf pipe and preserves liquid 300 μ l and seminal fluid to be saved 100 μ l, fully mixing, it is placed in-20 DEG C or-80 DEG C preservations, during detection, after its equilibrium at room temperature, adjust sperm concentration to 5～10 × 10 with normal saline6/ml；
3. detecting step
1) take off-the-shelf specimen to be measured 60 μ l, add in the sample cell that above-mentioned meltable gel has melted, fully mix, obtain suspension, hatch stand-by for 37 DEG C；
2) will be coated after microscope slide is placed in 2～8 DEG C of refrigerator pre-coolings 5 minutes and take out, and be coated region in microscope slide rapidly and add step 1) the suspension 30 μ l for preparing；
3) cover plate is covered rapidly, it is to avoid produce bubble；Put 2～8 DEG C of refrigerators 5 minutes so that it is solidification；
4) from refrigerator, take out microscope slide, carefully remove the superincumbent cover plate of covering；
5) being dipped vertically into by microscope slide in the reaction tank filling reactant liquor A immediately, 20～28 DEG C are reacted 7 minutes；
6) take out microscope slide, suck the liquid remaining in the microscope slide back side and lateral margin with filter paper；Microscope slide is dipped vertically in the reaction tank filling reactant liquor B, 20～28 DEG C of accurate responses 25 minutes；
7) take out microscope slide, suck the liquid remaining in the microscope slide back side and lateral margin with filter paper；By in the microscope slide level substantial amounts of purified water of immersion 5 minutes, change water therebetween 1～2 time；
8) take out microscope slide, suck the liquid remaining in the microscope slide back side and lateral margin with filter paper；Microscope slide is dipped vertically in the reaction tank filling 70% ethanol, 2 minutes；
9) take out microscope slide, suck the liquid remaining in the microscope slide back side and lateral margin with filter paper；Microscope slide is dipped vertically in the reaction tank filling 90% ethanol, 2 minutes；
10) take out microscope slide, suck the liquid remaining in the microscope slide back side and lateral margin with filter paper；Microscope slide is dipped vertically in the reaction tank filling 100% ethanol, 2 minutes；
11) natural drying in air；
12) every microscope slide is with Wright's stain 15～20 covering, is slowly added into Rui Shi buffer 30～40 again, blows and beats mixing dye liquor with ear washing bulb gently after waiting 1min, rinses dye sheet after room temperature standing 15min gently with flowing water；
13) natural drying or dry up；
14) 500 sperms of 40 × basis of microscopic observation, there is the sperm quantity of DNA fragment in counting.
Due to technique scheme, the present invention has following technical effect that
1, the present invention optimizes agent structure according to detection method so that stable reagent, reliable, and applies when these reagent detect simple to operate and without special detection instrument, it is simple to routine clinical application and popularization.
2, present invention employs exclusive SCD and preserve liquid, it is possible to effectively preserving semen sample, its DNA fragment rate does not change within-20 DEG C of frozen fortnights.So preserving liquid by SCD and can easily preserve required specimen, the carrying out do not limit by T/A detects, and saves substantial amounts of manpower and materials.
Accompanying drawing explanation
Fig. 1 is the concrete steps 4 of DNA fragment with spermatozoon detection kit detection) in promote forward the schematic diagram of cover plate gently along cover plate lower end.
Fig. 2 be DNA fragment with spermatozoon detection kit detection concrete steps 4) in pinch the jag of cover plate and take the schematic diagram of cover plate along microscope slide plane lightly away.
Detailed description of the invention
The invention provides a kind of DNA fragment with spermatozoon detection kit, including:
It is coated microscope slide；
Meltable gel；
A liquid；
B liquid；
Wright's stain；
Rui Shi buffer；
SCD preserves liquid.
The described microscope slide that is coated is for being coated with the microscope slide of the aqueous solution of 0.1～5% low melting point (fusing point is 20～60 DEG C) agarose；Described meltable gel (fusing point is 20～60 DEG C) is the aqueous solution containing 0.1～5% agarose；Described A liquid be containing 0.01%～0.5% sodium chloride, 0.1%～5% glycine, 0.1%～1% glacial acetic acid, 0.01%～0.5% hydrogen peroxide aqueous solution；Described B liquid is be the aqueous solution of 7.0～8.0 containing 0.1%～1%SDS, 1%～10%Tris, 0.1%～1% 2 water disodiumedetate, 0.1%～1% polysorbas20 and pH value；Described Wright's stain is the methanol solution containing 0.05～0.5% Rui Shi pigment, 0.01～0.1% Ji Shi pigment；Described Rui Shi buffer is 0.01～0.1mol/L phosphate buffer of pH value 6.0～7.0；It is the aqueous solution containing 0.1～1% sodium chloride, 40～80% polysorbas20s that described SCD preserves liquid.The detection of existing DNA fragment must use the semen sample of fresh semen specimen or Liquid nitrogen, when using fresh semen specimen, and inconvenient, the detection time is also restrained, consumes manpower and materials and also compares many；And when adopting Liquid nitrogen, the cost of preservation is then too high.And the SCD that the present invention uses preserves liquid and can preserve semen sample, its DNA fragment rate does not change within-20 DEG C of frozen fortnights.So preserving liquid by SCD and can easily preserve required specimen, the carrying out do not limit by T/A detects, for instance can make a collection of specimens, and detection in several days once, saves substantial amounts of manpower and materials.
Described be coated microscope slide preparation method be:
1) weighing low melting-point agarose 1～50g and be placed in the container that can load 1000ml, add 50～200ml purified water, 50 ± 5 DEG C of stirring in water bath make agarose be completely dissolved.
2) purified water constant volume is added to 1000ml, stirring and evenly mixing, 50 ± 5 DEG C 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) weighing 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 DEG C of stirring in water bath make agarose be completely dissolved.
2) purified water constant volume is added to 1000ml, stirring and evenly mixing, 50 ± 5 DEG C of water bath heat preservations 2 hours.
3) while hot by step 2) solution is sub-packed in 0.5ml sample cell, often pipe 0.14ml.
The preparation method of described A liquid is:
1) taking 50～200ml purified water to be placed in 1000ml volumetric flask, weigh sodium chloride 0.1～5g, glycine 1～50g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
2) measuring glacial acetic acid 1～10ml, 30% hydrogen peroxidase 10 .33～16.7ml, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
3) it is settled to 1000ml, stirring and evenly mixing with purified water.
The preparation method of described B liquid is:
1) take 50～200ml purified water and be placed in 1000ml volumetric flask, weigh SDS (sodium lauryl sulphate) 1～10g, Tris (trishydroxymethylaminomethane) 10～100g, two water disodiumedetate (producers: 1Guanghua Chemical Plant Co., Ltd., Guangdong, model: 250g/ bottle) 1～10g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
2) measuring polysorbas20 1～10ml, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
3) under pH detects, it is slowly added into concentrated hydrochloric acid, regulates pH to 7.0～8.0.
4) it is settled to 1000ml with purified water.
The preparation method of described Wright's stain is:
1) weigh 0.25～2.5g Rui Shi pigment, 0.05～0.5g Ji Shi pigment, put in clean beaker, add 5～20ml methanol, grind a moment, sucking-off upper strata dye liquor；Add 5 again～20ml methanol, continue to grind, then liquid in sucking-off, so continuously several times, until dye powder all dissolves；
2) with methanol constant volume to 500ml, stirring and evenly mixing, it is transferred to brown reagent bottle and preserves.
The preparation method of described Rui Shi buffer is:
1) taking 50～200ml purified water to be placed in 1000ml volumetric flask, weigh disodium hydrogen phosphate 1.071～10.712g, potassium dihydrogen phosphate 1.107～11.067g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
2) detecting solution ph with pH meter, 6.0～7.0 is qualified, meta-acid dense disodium hydrogen phosphate adjustment, meta-alkali potassium dihydrogen phosphate adjustment.
3) it is settled to 1000ml with purified water.
Described SCD preserves the preparation method of liquid:
1) taking 50～200ml purified water to be placed in 1000ml volumetric flask, weigh sodium chloride 1～10g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
2) measuring polysorbas20 400～800ml, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
3) it is settled to 1000ml, stirring and evenly mixing with purified water.
Below by specific embodiment, the present invention is described in further detail.
Embodiment 1
Present embodiments provide a kind of DNA fragment with spermatozoon detection kit, including:
It is coated microscope slide；
Meltable gel；
A liquid；
B liquid；
Wright's stain；
Rui Shi buffer；
SCD preserves liquid.
Described it is coated the microscope slide that microscope slide is the aqueous solution being coated with 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 chloride, 0.55% glycine, 0.25% glacial acetic acid and 0.045% hydrogen peroxide；Described B liquid is be the aqueous solution of 7.2～7.4 containing 0.25%SDS, 2.422%Tris, 0.125% 2 water disodiumedetate, 0.15% polysorbas20 and pH value；Described Wright's stain is the methanol solution containing 0.1% Rui Shi pigment, 0.03% Ji Shi pigment；Described Rui Shi buffer is the 0.03mol/L phosphate buffer of pH value 6.2～6.4；It is the aqueous solution containing 0.36% sodium chloride, 45% polysorbas20 that described SCD preserves liquid.Described SCD preserves liquid can preserve semen sample, and in-20 DEG C of frozen fortnights, its DNA fragment rate will not change.
Described be coated microscope slide preparation method be:
1) low melting-point agarose [producer: Sigma-Aldrich (Shanghai) trade Co., Ltd is weighed, model: 25g/ bottle] 5g is placed in the container that can load 1000ml, adding purified water 100ml, 50 ± 5 DEG C of stirring in water bath make agarose be completely dissolved.
2) purified water constant volume is added to 1000ml, stirring and evenly mixing, 50 ± 5 DEG C 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) agarose [producer: Sigma-Aldrich (Shanghai) trade Co., Ltd is weighed, model: 25g/ bottle] 2.5g puts beaker, being placed in the container that can load 1000ml, add purified water 100ml, in water bath, 50 ± 5 DEG C of stirring in water bath make agarose be completely dissolved.
2) purified water constant volume is added to 1000ml, stirring and evenly mixing, 50 ± 5 DEG C of water bath heat preservations 2 hours.
3) while hot by step 5) solution is sub-packed in 0.5ml sample cell, often pipe 0.14ml.
The preparation method of described A liquid is:
1) taking 100ml purified water to be placed in 1000ml volumetric flask, weigh sodium chloride 0.5g, glycine 5.5g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
2) measuring glacial acetic acid 2.5ml, 30% hydrogen peroxide 1.5ml, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
3) it is settled to 1000ml, stirring and evenly mixing with purified water.
The preparation method of described B liquid is:
1) taking 100ml purified water to be placed in 1000ml volumetric flask, weigh SDS2.5g, Tris24.22g, two water disodiumedetate 1.25g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
2) measuring polysorbas20 1.5ml, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
3) under pH detects, it is slowly added into concentrated hydrochloric acid, regulates pH to 7.2～7.4.
4) it is settled to 1000ml with purified water.
The preparation method of described Wright's stain is:
1) weigh 0.5g Rui Shi pigment, 0.15g Ji Shi pigment, put in clean beaker, add 10ml methanol, grind a moment, sucking-off upper strata dye liquor；Add 10ml methanol again, continue to grind, then liquid in sucking-off, so continuously several times, until dye powder all dissolves；
2) with methanol constant volume to 500ml, stirring and evenly mixing, it is transferred to brown reagent bottle and preserves.
The preparation method of described Rui Shi buffer is:
1) taking appropriate purified water to be placed in 1000ml volumetric flask, weigh disodium hydrogen phosphate 3.213g, potassium dihydrogen phosphate 3.320g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
2) detecting solution ph with pH meter, 6.2～6.4 is qualified, meta-acid dense 12 water disodium hydrogen phosphate adjustment, meta-alkali potassium dihydrogen phosphate adjustment.
3) it is settled to 1000ml with purified water.
Described SCD preserves the preparation method of liquid:
1) taking 100ml purified water to be placed in 1000ml volumetric flask, weigh sodium chloride 3.6g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
2) measuring polysorbas20 450ml, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
3) it is settled to 1000ml, stirring and evenly mixing with purified water.
Embodiment 2
Present embodiments provide a kind of DNA fragment with spermatozoon detection kit, including:
It is coated microscope slide；
Meltable gel；
A liquid；
B liquid；
Wright's stain；
Rui Shi buffer；
SCD preserves liquid.
Described it is coated the microscope slide that microscope slide is the aqueous solution being coated with 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 chloride, 2.2% glycine, 0.10% glacial acetic acid and 0.18% hydrogen peroxide；Described B liquid is be the aqueous solution of 7.6～8.0 containing 1% sodium lauryl sulphate (SDS), 10%Tris, 0.5% 2 water disodiumedetate, 0.6% polysorbas20 and pH value；Described Wright's stain is the methanol solution containing 0.4% Rui Shi pigment, 0.12% Ji Shi pigment；Described Rui Shi buffer is the 0.12mol/L phosphate buffer of pH value 6.8～7.0；Described SCD preserves the aqueous solution that liquid is 1.44% sodium chloride, 75% polysorbas20.Described SCD preserves liquid can preserve semen sample, and in-20 DEG C of frozen fortnights, its DNA fragment rate will not change.
Described be coated microscope slide preparation method be:
1) low melting-point agarose [producer: Sigma-Aldrich (Shanghai) trade Co., Ltd is weighed, model: 25g/ bottle] 20g is placed in the container that can load 1000ml, adding purified water 100ml, 50 ± 5 DEG C of stirring in water bath make agarose be completely dissolved.
2) purified water constant volume is added to 1000ml, stirring and evenly mixing, 50 ± 5 DEG C 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) agarose [producer: Sigma-Aldrich (Shanghai) trade Co., Ltd is weighed, model: 25g/ bottle] 10g puts beaker, being placed in the container that can load 1000ml, add purified water 100ml, in water bath, 50 ± 5 DEG C of stirring in water bath make agarose be completely dissolved.
2) purified water constant volume is added to 1000ml, stirring and evenly mixing, 50 ± 5 DEG C of water bath heat preservations 2 hours.
3) while hot by step 5) solution is sub-packed in 0.5ml sample cell, often pipe 0.14ml.
The preparation method of described A liquid is:
1) taking 100ml purified water to be placed in 1000ml volumetric flask, weigh sodium chloride 2g, glycine 22g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
2) measuring glacial acetic acid 1.0ml, 30% hydrogen peroxide 6.0ml, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
3) it is settled to 1000ml, stirring and evenly mixing with purified water.
The preparation method of described B liquid is:
1) taking 100ml purified water to be placed in 1000ml volumetric flask, weigh SDS10g, Tris100g, 2 water disodiumedetate 5g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
2) measuring polysorbas20 6.0ml, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
3) under pH detects, it is slowly added into concentrated hydrochloric acid, regulates pH to 7.6～8.0.
4) it is settled to 1000ml with purified water.
The preparation method of described Wright's stain is:
1) weigh 2.0g Rui Shi pigment, 0.6g Ji Shi pigment, put in clean beaker, add 10ml methanol, grind a moment, sucking-off upper strata dye liquor；Add 10ml methanol again, continue to grind, then liquid in sucking-off, so continuously several times, until dye powder all dissolves；
2) with methanol constant volume to 500ml, stirring and evenly mixing, it is transferred to brown reagent bottle and preserves.
The preparation method of described Rui Shi buffer is:
1) taking appropriate purified water to be placed in 1000ml volumetric flask, weigh disodium hydrogen phosphate 12.854g, potassium dihydrogen phosphate 13.28g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
2) detecting solution ph with pH meter, 6.8～7.0 is qualified, meta-acid dense 12 water disodium hydrogen phosphate adjustment, meta-alkali potassium dihydrogen phosphate adjustment.
3) it is settled to 1000ml with purified water.
Described SCD preserves the preparation method of liquid:
1) taking 100ml purified water to be placed in 1000ml volumetric flask, weigh sodium chloride 14.4g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
2) measuring polysorbas20 750ml, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
3) it is settled to 1000ml, stirring and evenly mixing with purified water.
Embodiment 3
Present embodiments provide a kind of DNA fragment with spermatozoon detection kit, including:
It is coated microscope slide；
Meltable gel；
A liquid；
B liquid；
Wright's stain；
Rui Shi buffer；
SCD preserves liquid.
Described it is coated the microscope slide that microscope slide is the aqueous solution being coated with 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 chloride, 1.1% glycine, 0.50% glacial acetic acid and 0.09% hydrogen peroxide；Described B liquid is be the aqueous solution of 7.3～7.7 containing 0.5% sodium lauryl sulphate (SDS), 4.844%Tris, 0.2525% 2 water disodiumedetate, 0.3% polysorbas20 and pH value；Described Wright's stain is the methanol solution containing 0.2% Rui Shi pigment, 0.06% Ji Shi pigment；Described Rui Shi buffer is the 0.06mol/L phosphate buffer of pH value 6.4～6.8；Described SCD preserves the aqueous solution that liquid is 0.72% sodium chloride, 65% polysorbas20.Described SCD preserves liquid can preserve semen sample, and in-20 DEG C of frozen fortnights, its DNA fragment rate will not change.
Described be coated microscope slide preparation method be:
1) low melting-point agarose [producer: Sigma-Aldrich (Shanghai) trade Co., Ltd is weighed, model: 25g/ bottle] 10g is placed in the container that can load 1000ml, adding purified water 100ml, 50 ± 5 DEG C of stirring in water bath make agarose be completely dissolved.
2) purified water constant volume is added to 1000ml, stirring and evenly mixing, 50 ± 5 DEG C 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) agarose [producer: Sigma-Aldrich (Shanghai) trade Co., Ltd is weighed, model: 25g/ bottle] 5g puts beaker, being placed in the container that can load 1000ml, add purified water 100ml, in water bath, 50 ± 5 DEG C of stirring in water bath make agarose be completely dissolved.
2) purified water constant volume is added to 1000ml, stirring and evenly mixing, 50 ± 5 DEG C of water bath heat preservations 2 hours.
3) while hot by step 5) solution is sub-packed in 0.5ml sample cell, often pipe 0.14ml.
The preparation method of described A liquid is:
1) taking 100ml purified water to be placed in 1000ml volumetric flask, weigh sodium chloride 1g, glycine 11g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
2) measuring glacial acetic acid 5.0ml, 30% hydrogen peroxide 3.0ml, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
3) it is settled to 1000ml, stirring and evenly mixing with purified water.
The preparation method of described B liquid is:
1) taking 100ml purified water to be placed in 1000ml volumetric flask, weigh SDS5g, Tris48.44g, 2 water disodiumedetate 2.525g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
2) measuring polysorbas20 3.0ml, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
3) under pH detects, it is slowly added into concentrated hydrochloric acid, regulates pH to 7.5 ± 0.2.
4) it is settled to 1000ml with purified water.
The preparation method of described Wright's stain is:
1) weigh 1.0g Rui Shi pigment, 0.3g Ji Shi pigment, put in clean beaker, add 10ml methanol, grind a moment, sucking-off upper strata dye liquor；Add 10ml methanol again, continue to grind, then liquid in sucking-off, so continuously several times, until dye powder all dissolves；
2) with methanol constant volume to 500ml, stirring and evenly mixing, it is transferred to brown reagent bottle and preserves.
The preparation method of described Rui Shi buffer is:
1) taking appropriate purified water to be placed in 1000ml volumetric flask, weigh disodium hydrogen phosphate 6.427g, potassium dihydrogen phosphate 6.640g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
2) detecting solution ph with pH meter, 6.4～6.8 is qualified, meta-acid dense 12 water disodium hydrogen phosphate adjustment, meta-alkali potassium dihydrogen phosphate adjustment.
3) it is settled to 1000ml with purified water.
Described SCD preserves the preparation method of liquid:
1) taking 100ml purified water to be placed in 1000ml volumetric flask, weigh sodium chloride 7.2g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
2) measuring polysorbas20 650ml, add in above-mentioned volumetric flask, stirring makes it be completely dissolved.
3) it is settled to 1000ml, stirring and evenly mixing with purified water.
Embodiment 4
Present embodiments provide specifically comprising the following steps that of DNA fragment with spermatozoon detection kit detection
1. reagent prepares
1) will be equipped with the sample cell of meltable gel (having the meltable gel of 0.14ml in pipe) to be placed in 80 DEG C and hatch 20 minutes, after melting completely, meltable gel tube is placed in 37 DEG C stand-by (at least balance 5 minutes).
2) before detection, room temperature is adjusted to about 20～28 DEG C.
2. specimen prepares
1) with the fresh spermatozoa of normal saline adjustment liquefaction (the sperm of liquid nitrogen cryopreservation or extracted after motile sperm) concentration is to 5～10 × 106/ml。
2) fresh specimens of detection can not be completed, it is possible to use freezen protective after SCD preservation liquid, can at least preserve 15 days.Method is as follows: adds SCD in Eppendorf pipe and preserves liquid 300 μ l and seminal fluid to be saved 100 μ l, fully mixes, be placed in-20 DEG C or-80 DEG C preservations.During detection, after its equilibrium at room temperature, adjust sperm concentration to 5～10 × 10 with normal saline6/ml。
3. detecting step
1) take off-the-shelf specimen to be measured 60 μ l, add in the sample cell that above-mentioned meltable gel has melted and (note 37 DEG C of persistently insulations), fully mix, obtain suspension, hatch stand-by for 37 DEG C.
2) will be coated after microscope slide (specification is 76.2 × 25.4mm) is placed in 2～8 DEG C of refrigerator pre-coolings 5 minutes and take out, and be coated region in microscope slide rapidly and add step 1) the suspension 30 μ l for preparing.
3) cover plate is covered rapidly, it is to avoid produce bubble；Put 2～8 DEG C of refrigerators 5 minutes so that it is solidification.
4) from refrigerator, take out microscope slide, carefully remove the superincumbent cover plate of covering.Method: as it is shown in figure 1, promote forward cover plate gently along cover plate lower end, until the cover plate other end is slightly beyond microscope slide width；Pinch the jag of cover plate, take cover plate lightly away along microscope slide plane, as shown in Figure 2.Noting: in the process of mobile cover plate, microscope slide planar slide is close to all the time by cover plate, and cover plate can't upwards be lifted away from gel plane.
5) being dipped vertically into by microscope slide in the reaction tank filling reactant liquor A (A liquid) immediately, 20～28 DEG C are reacted 7 minutes.
6) take out microscope slide, suck the liquid (not contact specimen district) remaining in the microscope slide back side and lateral margin with filter paper；Microscope slide is dipped vertically in the reaction tank filling reactant liquor B (B liquid), 20～28 DEG C of accurate responses 25 minutes.
7) take out microscope slide, suck the liquid (not contact specimen district) remaining in the microscope slide back side and lateral margin with filter paper；By in the microscope slide level substantial amounts of purified water of immersion 5 minutes, change water therebetween 1～2 time.
8) take out microscope slide, suck the liquid (not contact specimen district) remaining in the microscope slide back side and lateral margin with filter paper；Microscope slide is dipped vertically in the reaction tank filling 70% ethanol, 2 minutes.
9) take out microscope slide, suck the liquid (not contact specimen district) remaining in the microscope slide back side and lateral margin with filter paper；Microscope slide is dipped vertically in the reaction tank filling 90% ethanol, 2 minutes.
10) take out microscope slide, suck the liquid (not contact specimen district) remaining in the microscope slide back side and lateral margin with filter paper；Microscope slide is dipped vertically in the reaction tank filling 100% ethanol, 2 minutes；
11) natural drying in air.
12) dye sheet.Every microscope slide is with Wright's stain 15～20 covering, Deng being slowly added into Rui Shi buffer 30～40 after 1min again, blow and beat mixing dye liquor (noting not destroying the surface tension that dye liquor is formed) with ear washing bulb gently, after room temperature standing 15min, rinse dye sheet with flowing water gently.
13) natural drying or dry up.
14) 500 sperms of 40 × basis of 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 unilateral halation is less than the 1/3 of sperm head minimum diameter.

Claims (10)

1. a DNA fragment with spermatozoon detection kit, it is characterised in that including:
It is coated microscope slide,
Meltable gel,
A liquid,
B liquid,
Wright's stain,
Rui Shi buffer,
SCD preserves liquid；
The described microscope slide being coated the aqueous solution that microscope slide is the agarose that fusing point is 20～60 DEG C being coated with 0.1～5%；Described meltable gel is the aqueous solution of the agarose that fusing point is 20～60 DEG C containing 0.1～5%；Described A liquid be containing 0.01%～0.5% sodium chloride, 0.1%～5% glycine, 0.1%～1% glacial acetic acid, 0.01%～0.5% hydrogen peroxide aqueous solution；Described B liquid is be the aqueous solution of 7.0～8.0 containing 0.1%～1%SDS, 1%～10%Tris, 0.1%～1% 2 water disodiumedetate, 0.1%～1% polysorbas20 and pH value；Described Wright's stain is the methanol solution containing 0.05～0.5% Rui Shi pigment, 0.01～0.1% Ji Shi pigment；Described Rui Shi buffer is 0.01～0.1mol/L phosphate buffer of pH value 6.0～7.0；It is the aqueous solution containing 0.1～1% sodium chloride, 40～80% polysorbas20s that described SCD preserves liquid.
2. DNA fragment with spermatozoon detection kit according to claim 1, it is characterised in that described in be coated the preparation method of microscope slide and be:
1) weighing low melting-point agarose 1～50g and be placed in the container that can load 1000ml, add 50～200ml purified water, 50 ± 5 DEG C of stirring in water bath make agarose be completely dissolved；
2) purified water constant volume is added to 1000ml, stirring and evenly mixing, 50 ± 5 DEG C 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, it is characterised in that the preparation method of described meltable gel is:
1) weighing 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 DEG C of stirring in water bath make agarose be completely dissolved；
2) purified water constant volume is added to 1000ml, stirring and evenly mixing, 50 ± 5 DEG C of water bath heat preservations 2 hours；
3) while hot by step 2) solution is sub-packed in 0.5ml sample cell, often pipe 0.14ml.
4. DNA fragment with spermatozoon detection kit according to claim 1, it is characterised in that the preparation method of described A liquid is:
1) taking 50～200ml purified water to be placed in 1000ml volumetric flask, weigh sodium chloride 0.1～5g, glycine 1～50g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved；
2) measuring glacial acetic acid 1～10ml, 30% hydrogen peroxidase 10 .33～16.7ml, add in above-mentioned volumetric flask, stirring makes it be completely dissolved；
3) it is settled to 1000ml, stirring and evenly mixing with purified water.
5. DNA fragment with spermatozoon detection kit according to claim 1, it is characterised in that the preparation method of described B liquid is:
1) taking 50～200ml purified water to be placed in 1000ml volumetric flask, weigh SDS1～10g, Tris10～100g, two water disodiumedetate 1～10g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved；
2) measuring polysorbas20 1～10ml, add in above-mentioned volumetric flask, stirring makes it be completely dissolved；
3) under pH detects, it is slowly added into concentrated hydrochloric acid, regulates pH to 7.0～8.0；
4) it is settled to 1000ml with purified water.
6. DNA fragment with spermatozoon detection kit according to claim 1, it is characterised in that the preparation method of described Wright's stain is:
1) weigh 0.25～2.5g Rui Shi pigment, 0.05～0.5g Ji Shi pigment, put in clean beaker, add 5～20ml methanol, grind a moment, sucking-off upper strata dye liquor；Add 5 again～20ml methanol, continue to grind, then liquid in sucking-off, so continuously several times, until dye powder all dissolves；
2) with methanol constant volume to 500ml, stirring and evenly mixing, it is transferred to brown reagent bottle and preserves.
7. DNA fragment with spermatozoon detection kit according to claim 1, it is characterised in that the preparation method of described Rui Shi buffer is:
1) taking 50～200ml purified water to be placed in 1000ml volumetric flask, weigh disodium hydrogen phosphate 1.071～10.712g, potassium dihydrogen phosphate 1.107～11.067g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved；
2) detecting solution ph with pH meter, 6.0～7.0 is qualified, meta-acid dense disodium hydrogen phosphate adjustment, meta-alkali potassium dihydrogen phosphate adjustment；
3) it is settled to 1000ml with purified water.
8. DNA fragment with spermatozoon detection kit according to claim 1, it is characterised in that described SCD preserves the preparation method of liquid and is:
1) taking 50～200ml purified water to be placed in 1000ml volumetric flask, weigh sodium chloride 1～10g, add in above-mentioned volumetric flask, stirring makes it be completely dissolved；
2) measuring polysorbas20 400～800ml, add in above-mentioned volumetric flask, stirring makes it be completely dissolved；
3) it is settled to 1000ml, stirring and evenly mixing with purified water.
9. DNA fragment with spermatozoon detection kit according to claim 1, it is characterised in that described in be coated the microscope slide that microscope slide is the aqueous solution being coated with 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 chloride, 0.55% glycine, 0.25% glacial acetic acid and 0.045% hydrogen peroxide；Described B liquid is be the aqueous solution of 7.2～7.4 containing 0.25%SDS, 2.422%Tris, 0.125% 2 water disodiumedetate, 0.15% polysorbas20 and pH value；Described Wright's stain is the methanol solution containing 0.1% Rui Shi pigment, 0.03% Ji Shi pigment；Described Rui Shi buffer is the 0.03mol/L phosphate buffer of pH value 6.2～6.4；It is the aqueous solution containing 0.36% sodium chloride, 45% polysorbas20 that described SCD preserves liquid.
10. the detection method of DNA fragment with spermatozoon detection kit according to claim 1, comprises the steps:
1. reagent prepares
1) will be equipped with the sample cell of the meltable gel of 0.14ml to be placed in 80 DEG C and hatch 20 minutes, after melting completely, meltable gel tube is placed in 37 DEG C stand-by, at least balance 5 minutes；
2) before detection, room temperature is adjusted to 20～28 DEG C；
2. specimen prepares
1) with normal saline adjustment liquefaction fresh spermatozoa or liquid nitrogen cryopreservation sperm or extracted after motile sperm concentration to 5～10 × 106/ml；
2) fresh specimens of detection can not be completed, freezen protective after use SCD preservation liquid, method is as follows: adds SCD in Eppendorf pipe and preserves liquid 300 μ l and seminal fluid to be saved 100 μ l, fully mixing, it is placed in-20 DEG C or-80 DEG C preservations, during detection, after its equilibrium at room temperature, adjust sperm concentration to 5～10 × 10 with normal saline6/ml；
3. detecting step
1) take off-the-shelf specimen to be measured 60 μ l, add in the sample cell that above-mentioned meltable gel has melted, fully mix, obtain suspension, hatch stand-by for 37 DEG C；
2) will be coated after microscope slide is placed in 2～8 DEG C of refrigerator pre-coolings 5 minutes and take out, and be coated region in microscope slide rapidly and add step 1) the suspension 30 μ l for preparing；
3) cover plate is covered rapidly, it is to avoid produce bubble；Put 2～8 DEG C of refrigerators 5 minutes so that it is solidification；
4) from refrigerator, take out microscope slide, carefully remove the superincumbent cover plate of covering；
5) being dipped vertically into by microscope slide in the reaction tank filling A liquid immediately, 20～28 DEG C are reacted 7 minutes；
6) take out microscope slide, suck the liquid remaining in the microscope slide back side and lateral margin with filter paper, microscope slide is dipped vertically in the reaction tank filling B liquid, 20～28 DEG C of accurate responses 25 minutes；
7) take out microscope slide, suck the liquid remaining in the microscope slide back side and lateral margin with filter paper, by the microscope slide level substantial amounts of purified water of immersion 5 minutes, change water therebetween 1～2 time；
8) take out microscope slide, suck the liquid remaining in the microscope slide back side and lateral margin with filter paper, microscope slide is dipped vertically in the reaction tank filling 70% ethanol, 2 minutes；
9) take out microscope slide, suck the liquid remaining in the microscope slide back side and lateral margin with filter paper, microscope slide is dipped vertically in the reaction tank filling 90% ethanol, 2 minutes；
10) take out microscope slide, suck the liquid remaining in the microscope slide back side and lateral margin with filter paper, microscope slide is dipped vertically in the reaction tank filling 100% ethanol, 2 minutes；
11) natural drying in air；
12) every microscope slide is with Wright's stain 15～20 covering, is slowly added into Rui Shi buffer 30～40 again, blows and beats mixing dye liquor with ear washing bulb gently after waiting 1min, rinses dye sheet after room temperature standing 15min gently with flowing water；
13) natural drying or dry up；
14) 500 sperms of 40 × basis of microscopic observation, there is the sperm quantity of DNA fragment in counting.
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