CN102051413A - Human sperm DNA fragment detection method and detection kit - Google Patents

Abstract

The invention provides a human sperm DNA fragment detection method comprising the following steps: (1) diluting liquefied sperm with a buffer solution of 4 DEG C; (2) adding the diluted sperm into a sample tube of a flow cytometry; (3) adding an acid treatment buffer solution; (4) after 30 seconds, adding an AO staining solution; (5) setting a calibration and flow chamber of the flow cytometry, equilibrating a sample line by an AO equilibration buffer solution; (6) continuously determining each sample at least twice, recording at least 5000 cells and carrying out statistical analysis; and (7) after determining, respectively thoroughly cleaning the cell fragments and fluorescent dye remaining in the sample line of the flow cytometry by bleacher, sample tube detergent and degerming double distilled water. A human sperm DNA fragment detection kit comprises 50mL of staining buffer solution, 300mu L of AO stock solution, 20mL of acid treatment solution, 100mL of buffer solution, 100mL of sheath fluid, 100mL of AO remover, 100mL of sample tube detergent and 100mL of double distilled water (ddH2O). The invention realizes the purpose of clinically detecting the sperm DNA fragments simply, conveniently, quickly and accurately.

Description

A kind of human sperm DNA fragment detection method and detection kit

Technical field

The reagent that the present invention relates to use on the clinical medicine, particularly a kind of human sperm DNA fragment detection method and detection kit.

Background technology

Raising along with expanding economy and medical level, infertile sickness rate shows a rising trend on the contrary, a nowadays influence human lives and a healthy class principal disease have been become, wherein in China, the sickness rate of infertility is more than 10%, male factor and female factors respectively account for 30%, and both sides' common factor accounts for 40%.The sperm reason ranks first in the male factor, the genetic material carrier-DNA integrity of sperm can have influence on the fertility of sperm, the division of zygote and embryo's normal development, and that the DNA fragment with spermatozoon meeting causes is sterile, habitual abortion and help pregnant treatment success ratio low etc.Therefore the structural analysis of staining of sperm matter (DNA) is to detect sperm to be subjected to essence function, to judge one of extremely important lab index of fetal development, at present its tool authority's detection method is staining of sperm matter structure detection (Sperm Chormasome Str μ ct μ re Assay, be called for short SCSA), i.e. the advantage combination of acridine orange fluorescent dye and flow cytometry.Utilize the unusual sperm of chromatin to be subject to acid or thermally denature becomes single stranded DNA, combine rubescent look fluorescence with the dyestuff acridine orange; And the dna double chain structure that normochromatic sperm can be kept perfectly combines this characteristic of green-emitting fluorescence with acridine orange.Sample if the red values ratio increases, just illustrates that chromatin Structure increases unusually through after the denaturing treatment.

At present domesticly also be in the exploratory stage for the mechanism of DNA fragment with spermatozoonization and the treatment of DNA fragmentation, set up a kind of simple, standardized clinical detection method and detection kit so press for very much, simultaneously for setting up the stdn of compatriots' DNA fragment with spermatozoon trace routine and the stdn of compatriots' evaluation of result lays the foundation.

Summary of the invention

At the problem in the background technology, the object of the invention is to provide a kind of human sperm's DNA detection method and detection kit, has comprised that in test kit SCSA detects needed whole reagent.

For achieving the above object, by method and step experiment once:

A kind of human sperm DNA fragment detection method is characterized in that, comprises the steps:

1) Ye Hua seminal fluid is with 4 ℃ damping fluid dilution sperm to 1～2x10 ⁶Individual/mL;

2) add sperm that 200 μ L diluted in flow cytometer sample introduction pipe;

3) buffer solution for acid of adding 400 μ L;

4) after 30 seconds, add 1.2mL AO staining fluid;

5) calibration and the flow chamber of setting flow cytometer are with AO level pad balance sample wire;

6) each sample METHOD FOR CONTINUOUS DETERMINATION twice at least writes down 5000 cells at least and is write down and statistical study;

7) after the mensuration, thoroughly remove cell relic and the fluorescence dye that residues in the flow cytometer sample introduction line with the distilled water of SYNTHETIC OPTICAL WHITNER, sample introduction pipe sanitising agent and degerming respectively.

As preferred version, the damping fluid in the described step 1) is 0.01M Tris-HCl, 0.15M NaCl, and 1mM EDTA mixture, pH value are 7.4;

Buffer solution for acid in the described step 3) is 0.08N HCl, 0.15M NaCl, and the mixture of 0.1%Triton-X 100, pH value are 1.2.

The 1.2mLAO staining fluid is the 0.1M citric acid in the described step 4), 0.2M Na ₂HPO ₄, 1mM EDTA, 0.15M NaCl, the mixture of 6 μ g/mL acridine oranges;

The AO level pad is the mixture of 400 μ L pickling agents and 1.2mL AO staining fluid in the described step 5);

SYNTHETIC OPTICAL WHITNER in the described step 7) is 50% distilled water ddH ₂O and 50% the mixture that contains 5% clorox approximately, described sample introduction pipe sanitising agent is the mixture of 50% ethanol, 50% SYNTHETIC OPTICAL WHITNER and 0.5MNaCl.

Be the technical solution problem, the present invention returns provides a kind of detection kit, and it comprises: dyeing damping fluid 50mL, AO storing solution 300 μ L, pickling agent 20mL, damping fluid 100mL, sheath fluid 100mL, AO remove liquid 100mL, sample introduction pipe sanitising agent 100mL, distilled water (ddH ₂O) 100mL.

Preferably, described detection kit need dispose 0.1M citrate buffer solution and two kinds of basic reagent of 0.2M Na2HPO4 damping fluid: wherein the 0.1M citrate buffer solution adds to 1L ddH by the 21.01g citric acid ₂O is settled to 1L, makes for 4 ℃; 0.2M Na ₂HPO ₄Damping fluid is by 28.4g Na ₂HPO ₄Add to 1L ddH ₂O makes for 4 ℃.

Preferably, described dyeing damping fluid making method is: the Na of 370mL 0.1M citrate buffer solution, 630mL0.2M ₂HPO ₄Damping fluid, 372mg EDTA2Na and 8.77g NaCl, mixed solution stir and spend the night, and strip NaOH transfers pH value to 6.0;

Described pickling agent making method is: at the ddH of 300ml ₂O adds Triton X-100, the adding 20ml 2.0N HCl of 4.39g NaCl, 0.5mL, and distilled water is settled to 500mL, regulates PH to 1.2 with 5N HCl;

Described damping fluid collocation method is: take by weighing 9.48g Tris-HCl, 52.6gNaCl and 2.23gEDTA2Na, add 600ml ddH ₂O, stirring and evenly mixing, 2N NaOH regulates pH value to 7.4, dilutes 10 times then;

Preferably, described AO storing solution making method is: the AO powder of 5mg is dissolved in 5mL ddH2O, and the aluminium-foil paper parcel keeps in Dark Place;

Described AO staining fluid making method is: the AO storing solution of drawing 600 μ l earlier adds in the dyeing damping fluid of 100mL, the AO storing solution is added to the dyeing damping fluid again and blows and beats Tip afterwards repeatedly for several times, places brown bottle at last;

Described AO removes the liquid collocation method: with 50mL ddH ₂O and 50mL SYNTHETIC OPTICAL WHITNER (containing 5% clorox approximately), thorough mixing, room temperature preservation;

Preferably, described sheath fluid collocation method is: add 0.5ml Triton-X100 in every premium on currency, 0.45 μ m filter filters ddH ₂O;

Described sample introduction pipe sanitising agent collocation method is: thorough mixing 50%ETOH, 50% SYNTHETIC OPTICAL WHITNER (containing 5% clorox approximately) and 0.5MNaCl (2.29g/100mL);

Described G liquid is distilled water (ddH ₂O).

The beneficial effect that detection method of the present invention and detection kit are brought is:

1. a kind of detection method and detection kit of human sperm DNA fragment are provided.

2. method that provides and detection kit are more stable with respect to traditional its SCSA detected result of sperm check and analysis method.

3.SCSA what weigh is the integrity of parental gene group, does not need to come analyzing samples by opticmicroscope, so detect with respect to other expensive sperm qualities, SCSA has simply, quick, advantage that cost is low.

4. the detection popularized more of this test kit is meticulousr, more accurate, is compatriots' DNA fragment with spermatozoon standardized testing, and the redefining to set up and lay a good foundation of healthy sperm.

5. this test kit then detects at SCSA fully, and with loaded down with trivial details easy and simple to handleization of original complexity, commercialization has also guaranteed authority and accuracy that SCSA detects simultaneously.

Embodiment

Below in conjunction with accompanying drawing preferred embodiment of the present invention is described in detail, thereby protection scope of the present invention is made more explicit defining so that advantages and features of the invention can be easier to be it will be appreciated by those skilled in the art that.

Among the embodiment, following shortenings is represented following meaning, and the shortenings that does not add definition uses its common acceptable definition:

℃=degree centigrade; Min=minute; Sec=second; The M=mole; The mM=mmole; The mL=milliliter; μ L=microlitre.

Preparation before the experiment:

(1) reagent preparation

1), 0.1M citrate buffer solution: the 21.01g citric acid adds to 1L ddH ₂O is settled to 1L, but 4 ℃ of poke moons;

2), 0.2M Na ₂HPO ₄Damping fluid: 28.4g Na ₂HPO ₄Add to 1L ddH ₂O can preserve the several months for 4 ℃;

Be the reduction language tissue, the partial confounding compound adopts self-defining title, and is as follows:

3), A1 liquid (PH6.0 dye damping fluid):

(0.1M citric acid, 0.2M Na ₂HPO ₄, 1mM EDTA, 0.15M NaCl): the Na of 370mL 0.1M citrate buffer solution, 630mL 0.2M ₂HPO ₄Damping fluid, 372mg EDTA2Na and 8.77gNaCl, mixed solution stir and spend the night, and strip NaOH transfers pH value to 6.0, can preserve the several months for 4 ℃;

4), A2 liquid (AO storing solution):

The AO powder of 5mg is dissolved in 5mL ddH ₂O, the aluminium-foil paper parcel keeps in Dark Place, and can preserve the several months for 4 ℃;

5), A liquid (AO staining fluid):

A. the AO storing solution of drawing 600 μ l adds in the dyeing damping fluid of 100mL;

The b.AO storing solution is blown and beaten Tip for several times after adding to the dyeing damping fluid repeatedly;

C. place brown bottle, can preserve fortnight for 4 ℃;

6), B liquid (PH 1.2 pickling agents) (0.08N HCl, 0.15M NaCl, 0.1%Tri ton-X100):

DdH at 300ml ₂O adds Triton X-100, the adding 20ml2.0N HCl of 4.39g NaCl, 0.5mL, and distilled water is settled to 500mL, regulates PH to 1.2 with 5N HCl, can preserve the several months for 4 ℃;

7), C liquid (1x TNE buffer solution ph 7.4):

Take by weighing 9.48g Tri s-HCl, 52.6gNaCl and 2.23gEDTA2Na, add 600ml ddH ₂O, stirring and evenly mixing, 2N NaOH regulates pH value to 7.4, dilutes 10 times then, can preserve 1 year for 4 ℃;

8), D liquid (sheath fluid 0.05%Triton-X 100):

Add 0.5ml Triton-X100 in every premium on currency, 0.45 μ m filter filters ddH ₂O;

9), E liquid (AO removes liquid):

With 50mL ddH ₂O and 50mL SYNTHETIC OPTICAL WHITNER (containing 5% clorox approximately), thorough mixing, room temperature preservation;

10), F liquid (sample introduction pipe sanitising agent):

Thorough mixing 50%ETOH, 50% SYNTHETIC OPTICAL WHITNER (containing 5% clorox approximately) and 0.5MNaCl (2.29g/100mL), room temperature preservation.

11), G liquid: distilled water (ddH ₂O).

Test kit of the present invention contains: A1 liquid 50mL, A2 liquid 300 μ L, B liquid 20mL, C liquid 100mL, D liquid 100mL, E liquid 100mL, F liquid 100mL, G liquid 100mL; And all reagent can both satisfy 10 person-times SCSA detection.

(2) A2 liquid is all joined in the A1 liquid, blow and beat repeatedly for several times, obtain A liquid (A liquid can be preserved fortnight at 4 ℃, and bottle cap is loaded onto 0.8～3.0mL and can be regulated the hand nozzle during use) with Tip.

The cryopreservation sperm cell:

After the sperm sample took out, room temperature was placed 30min, made sperm liquefaction;

Measure sperm concentration;

With C liquid rapid dilution sperm, making sperm concentration is 1～2x10 ⁶Individual/mL;

The semen sample that dilution is good does not add freezing preservatives, immediately in-70 ℃～-100 ℃ frozen, or liquid nitrogen cryopreservation.Sample to be measured places frozen pipe, and the liquid level and the mouth of pipe leave 1/4～1/2 space of sample volume in the pipe, to reduce active oxygen to the damage of sample (keep frozen pipe vertically to place when freezing, liquid sample froze at the pipe end, and water-bath is thawed and is more prone to like this) as far as possible.

(3) setting of the calibration of flow cytometer and flow chamber:

1) checks the instrument of calibration instrument with standardized fluorescent bead.The D liquid that in the sheath fluid bucket, adds 4/5 volume.

2) before the calibration flow cytometer, get 400 μ L B liquid to the sample introduction pipe, add the A liquid of 1.20mL behind the 30sec again, in sample wire, move 15min, with balance sample introduction pipe with the hand the regulated nozzle pressure of 0.2～0.8mL.

Setting suitable liquid stream condition, is 1.5x10 to several concentration tentatively ⁶The sample of individual/mL is measured, and regulates the flow velocity of cell, makes the flow velocity of sperm reach 200/sec, during the application cell screening instrument, uses cell low speed could obtain correct result.

(4) determination step

1) preparation of cell and dyeing:

1. take out frozen sample, place 37 ℃ of water-baths to thaw immediately, the firm thawing of ice cube will be taken out immediately.

2. in the sample introduction pipe of flow cytometer, add the spermatid that 200 μ L concentration are 1～2x106/mL.

3. the B liquid (cell places operation on ice) that adds 400 μ L with nozzle.

4. after the accurate timing 30Sec, add 1.2mL A liquid.

2) flow cytometer test sample:

1. will dye lustful sample and add the sample chamber, sample should start sample flow after entering the sample chamber immediately.

2. pick up counting immediately with stopwatch, the sampled data time for reading is set at 3min (detect the velocity of flow of sperm, if speed is too fast, cell flow rate just should be used C liquid dilute sample again greater than 300/second).

3. replication sample (each sample is METHOD FOR CONTINUOUS DETERMINATION twice at least, will have the measured value of 5000 cells to be write down and add up at least).

4. each sample is measured after the end for the second time, gets 400 μ L B liquid to the sample introduction pipe with 0 nozzle pressure in well, adds the A liquid of 1.20ml behind the 30sec again, the balance sample wire.

5. according to above step, continue to measure next sample (every day or measure different samples after all will finely tune).

6. after SCSA measured and finishes, the staining fluid in the sample wire was removed clean with E liquid.

7. F liquid moves 10min in sample wire, is used for thoroughly removing cell relic and some fluorescence dyes that residue in flow cytometer sample introduction line.

8. use G liquid at last again, clean sample wire.

The above; it only is the specific embodiment of the present invention; but protection scope of the present invention is not limited thereto; any those of ordinary skill in the art are in the disclosed technical scope of the present invention; variation or the replacement that can expect without creative work all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain that claims were limited.

Claims (7)

1. a human sperm DNA fragment detection method is characterized in that, comprises the steps:

1) Ye Hua seminal fluid is with 4 ℃ damping fluid dilution sperm to 1～2x10 ⁶Individual/mL;

2) add sperm that 200 μ L diluted in flow cytometer sample introduction pipe;

3) buffer solution for acid of adding 400 μ L;

4) after 30 seconds, add 1.2mL AO staining fluid;

5) calibration and the flow chamber of setting flow cytometer are with AO level pad balance sample wire;

6) each sample METHOD FOR CONTINUOUS DETERMINATION twice at least writes down 5000 cells at least and is write down and statistical study;

7) after the mensuration, thoroughly remove cell relic and the fluorescence dye that residues in the flow cytometer sample introduction line with the distilled water of SYNTHETIC OPTICAL WHITNER, sample introduction pipe sanitising agent and degerming respectively.

2. a kind of human sperm DNA fragment detection method according to claim 1 is characterized in that,

Damping fluid in the described step 1) is 0.01M Tris-HCl, 0.15M NaCl, and 1mM EDTA mixture, pH value are 7.4;

Buffer solution for acid in the described step 3) is 0.08N HCl, 0.15M NaCl, and the mixture of 0.1%Triton-X100, pH value are 1.2.

The 1.2mLAO staining fluid is the 0.1M citric acid in the described step 4), 0.2M Na ₂HPO ₄, 1mM EDTA, 0.15M NaCl, the mixture of 6 μ g/mL acridine oranges;

The AO level pad is the mixture of 400 μ L pickling agents and 1.2mL AO staining fluid in the described step 5);

SYNTHETIC OPTICAL WHITNER in the described step 7) is 50% distilled water ddH ₂O and 50% the mixture that contains 5% clorox approximately, described sample introduction pipe sanitising agent is the mixture of 50% ethanol, 50% SYNTHETIC OPTICAL WHITNER and 0.5MNaCl.

3. human sperm DNA fragment detection kit, it is characterized in that it comprises: dyeing damping fluid 50mL, AO storing solution 300 μ L, pickling agent 20mL, damping fluid 100mL, sheath fluid 100mL, AO remove liquid 100mL, sample introduction pipe sanitising agent 100mL, distilled water (ddH ₂O) 100mL.

4. a kind of human sperm DNA fragment detection kit according to claim 3 is characterized in that described detection kit need dispose 0.1M citrate buffer solution and 0.2M Na ₂HPO ₄Two kinds of basic reagent of damping fluid:

Wherein the 0.1M citrate buffer solution adds to 1L ddH by the 21.01g citric acid ₂O is settled to 1L, makes for 4 ℃; 0.2M Na ₂HPO ₄Damping fluid adds to 1L ddH by 28.4g Na2HPO4 ₂O makes for 4 ℃.

5. a kind of human sperm DNA fragment detection kit according to claim 3 is characterized in that,

Described dyeing damping fluid making method is: the Na of 370mL 0.1M citrate buffer solution, 630mL 0.2M ₂HPO ₄Damping fluid, 372mg EDTA2Na and 8.77g NaCl, mixed solution stir and spend the night, and strip NaOH transfers pH value to 6.0;

Described pickling agent making method is: at the ddH of 300ml ₂O adds Triton X-100, the adding 20ml 2.0N HCl of 4.39g NaCl, 0.5mL, and distilled water is settled to 500mL, regulates PH to 1.2 with 5N HCl;

Described damping fluid collocation method is: take by weighing 9.48g Tris-HCl, 52.6gNaCl and 2.23gEDTA2Na, add 600ml ddH ₂O, stirring and evenly mixing, 2N NaOH regulates pH value to 7.4, dilutes 10 times then;

6. a kind of human sperm DNA fragment detection kit according to claim 3 is characterized in that,

Described AO storing solution making method is: the AO powder of 5mg is dissolved in 5mL ddH ₂O, the aluminium-foil paper parcel keeps in Dark Place;

Described AO staining fluid making method is: the AO storing solution of drawing 600 μ l earlier adds in the dyeing damping fluid of 100mL, the AO storing solution is added to the dyeing damping fluid again and blows and beats Tip afterwards repeatedly for several times, places brown bottle at last;

Described AO removes the liquid collocation method: with 50mL ddH ₂O and 50mL SYNTHETIC OPTICAL WHITNER (containing 5% clorox approximately), thorough mixing, room temperature preservation;

7. a kind of human sperm DNA fragment detection kit according to claim 3 is characterized in that,

Described sheath fluid collocation method is: add 0.5ml Triton-X100 in every premium on currency, 0.45 μ m filter filters ddH ₂O;

Described sample introduction pipe sanitising agent collocation method is: thorough mixing 50%ETOH, 50% SYNTHETIC OPTICAL WHITNER (containing 5% clorox approximately) and 0.5MNaCl (2.29g/100mL);

Described G liquid is distilled water (ddH ₂O).