CN113432959A - Preparation method of quality control product for sperm DNA fragmentation detection - Google Patents
Preparation method of quality control product for sperm DNA fragmentation detection Download PDFInfo
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- CN113432959A CN113432959A CN202110555669.7A CN202110555669A CN113432959A CN 113432959 A CN113432959 A CN 113432959A CN 202110555669 A CN202110555669 A CN 202110555669A CN 113432959 A CN113432959 A CN 113432959A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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Abstract
The invention discloses a preparation method of a quality control product for sperm DNA fragmentation detection, which relates to the field of sperm DFI quality control products and comprises the following components used in the process of testing the sperm DFI quality control product: 500 ml-1000 ml of chicken erythrocyte liquid, 150 ml-500 ml of MACS solution, 100 ml-200 ml of hydrogen peroxide with the concentration of 25% -30% and 150 ml-500 ml of MACS solution containing cell protection solution, and has the following beneficial effects: the method adopts chicken erythrocyte to simulate sperm cells, treats the chicken erythrocyte with hydrogen peroxide to fragment DNA of the chicken erythrocyte, can control the value of DFI by mixing with normal cells in a certain proportion, uses cell protection components to ensure that the cells are stored for a long time at the conventional storage temperature, and the DFI value is not influenced by the storage condition.
Description
Technical Field
The invention relates to the field of sperm DFI quality control products, in particular to a preparation method of a quality control product for sperm DNA fragmentation detection.
Background
The sperm DFI detection is an abbreviation of sperm DNA fragmentation detection, is a detection process related to sperm quality, the sperm DNA fragmentation degree is considered as a new index for evaluating the sperm quality and predicting fertility, and the sperm DFI detection by adopting a flow cytometer is widely applied to clinic.
But sperm DFI detect reagent is different in performance on the market, and the testing result can't accurate assurance, and some labs adopt real sperm sample as the inside quality control in the laboratory, and the problem that exists has: the quality control can not be carried out in a laboratory, liquid nitrogen is required for storage, the DFI value can not be controlled, and the large-scale clinical popularization can not be realized.
Disclosure of Invention
Based on the above, the present invention aims to provide a method for preparing a quality control material for detecting sperm DNA fragmentation, so as to solve the problems in the background art.
In order to achieve the purpose, the invention provides the following technical scheme: a preparation method of quality control products for sperm DNA fragmentation detection comprises the following components used in a sperm DFI quality control product test process: 500ml to 1000ml of chicken erythrocyte liquid, 150ml to 500ml of MACS solution, 100ml to 200ml of hydrogen peroxide with the concentration of 25 percent to 30 percent and 150ml to 500ml of MACS solution containing cell protection solution.
By adopting the technical scheme, the reagent kit has the advantages of being convenient to use, convenient to store, stable in result, capable of monitoring the performance of the reagent, convenient to use clinically and the like.
The invention is further provided with a preparation method of the sperm DFI quality control product, which comprises the following steps:
a1, centrifuging 500-1000 ml of chicken erythrocyte liquid by using a centrifuge for 5-10 min, setting the temperature at 10-40 ℃ and the lifting rate at 5-9 times;
a2, discarding the supernatant after centrifugation, adding 150 ml-500 ml of MACS solution, uniformly mixing and resuspending, dividing the chicken red blood cells into two parts and centrifuging;
a3, centrifuging one part of the mixture, removing the supernatant, adding 150 ml-500 ml of MACS solution containing cell protection solution into a centrifuge tube, mixing uniformly, and storing in a refrigerator at the temperature of 8 ℃ below zero for later use;
a4, opening an oven at 20-25 ℃ for standby, centrifuging the other part, discarding the supernatant, adding 150-500 ml of MACS solution into a centrifuge tube for resuspension, uniformly mixing, adding 100-200 ml of 30% hydrogen peroxide suspension, immediately placing the mixture into the oven at 20-25 ℃ for reaction for 1-2 hours, and paying attention to the fact that the cover of the centrifuge tube is not screwed too tightly;
a5, after the reaction is finished, taking out the centrifuge tube for centrifugation, discarding the supernatant, adding 150 ml-500 ml of MACS solution for cleaning for 3 times, finally resuspending by using 150 ml-500 ml of MACS solution containing cell protection solution, and transferring to a new centrifuge tube for later use;
a6, counting two groups of cells by flow cytometry or microscope, can be prepared into cell mixture with DFI value of 0% -100%.
Through adopting above-mentioned technical scheme, through adopting chicken erythrocyte to simulate sperm cell, through hydrogen peroxide treatment chicken erythrocyte makes its DNA fragmentation, mixes through certain proportion and normal cell, steerable DFI's value uses cell protection component to let the cell preserve for a long time under conventional storage temperature to the DFI value does not receive the influence of storage condition, has convenient to use, and it is convenient to store, and the result is stable, monitoring reagent performance, the progressive part such as clinical use of being convenient for.
The invention is further configured that each of the two chicken red blood cells in the step A2 has 200 ml-500 ml, the chicken red blood cell liquid is blood of SPF chicken or cock, and a certain anticoagulant is added during blood collection.
By adopting the technical scheme, when the chicken red blood cells are used, the coagulation of the red blood cells in the manufacturing process can be prevented, and the manufacturing effect is influenced.
The invention is further configured that the centrifuge tube used in the manufacturing step is a round-bottom centrifuge tube, the centrifuge tube needs to be slowly turned upside down when being centrifuged, the supernatant is discarded until the supernatant is cool and transparent, and the specific operation of resuspension is to resuspend the centrifuged cells, active substances and the like by using a proper MACS solution.
Through adopting above-mentioned technical scheme, round bottom centrifuging tube during the use, is favorable to preventing the adhesion of chicken red blood cell in the bottom of centrifuging tube, and is comparatively convenient when clean.
The invention is further provided that the components of the MACS solution containing the cell protection solution comprise 130ml to 480ml of the MACS solution, 3ml to 5ml of glycerol and 1ml to 3ml of trehalose.
By adopting the technical scheme, the glycerol and the trehalose are favorable for preventing the loss of cells during the centrifugal process of manufacturing and influencing the manufacturing effect.
In summary, the invention mainly has the following beneficial effects: has the advantages of convenient use, convenient storage, stable result, reagent performance monitoring, convenient clinical use and the like.
According to the invention, the chicken red blood cells are adopted to simulate sperm cells, the chicken red blood cells are treated by hydrogen peroxide to fragment DNA, the DFI value can be controlled by mixing with normal cells according to a certain proportion, the cells are stored for a long time at a conventional storage temperature by using cell protection components, and the DFI value is not influenced by storage conditions.
Drawings
FIG. 1 is a process flow diagram of the present invention.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
The following describes an embodiment of the present invention based on its overall structure.
A preparation method of quality control product for sperm DNA fragmentation detection is disclosed as shown in figure 1, and comprises the following components used in the sperm DFI quality control product test process: 500 ml-1000 ml chicken red blood cell liquid, 150 ml-500 ml MACS solution, 100 ml-200 ml hydrogen peroxide with concentration of 25% -30% and 150 ml-500 ml MACS solution containing cell protection liquid, the preparation method of the sperm DFI quality control product comprises the following steps: a1, centrifuging 500-1000 ml of chicken erythrocyte liquid by using a centrifuge for 5-10 min, setting the temperature at 10-40 ℃ and the lifting rate at 5-9 times;
a2, discarding the supernatant after centrifugation, adding 150 ml-500 ml of MACS solution, uniformly mixing and resuspending, dividing the chicken red blood cells into two parts and centrifuging;
a3, centrifuging one part of the mixture, removing the supernatant, adding 150 ml-500 ml of MACS solution containing cell protection solution into a centrifuge tube, mixing uniformly, and storing in a refrigerator at the temperature of 8 ℃ below zero for later use;
a4, opening an oven at 20-25 ℃ for standby, centrifuging the other part, discarding the supernatant, adding 150-500 ml of MACS solution into a centrifuge tube for resuspension, uniformly mixing, adding 100-200 ml of 30% hydrogen peroxide suspension, immediately placing the mixture into the oven at 20-25 ℃ for reaction for 1-2 hours, and paying attention to the fact that the cover of the centrifuge tube is not screwed too tightly;
a5, after the reaction is finished, taking out the centrifuge tube for centrifugation, discarding the supernatant, adding 150 ml-500 ml of MACS solution for cleaning for 3 times, finally resuspending by using 150 ml-500 ml of MACS solution containing cell protection solution, and transferring to a new centrifuge tube for later use;
a6, counting two groups of cells by a flow cytometer or a microscope, and preparing a cell mixture with a DFI value of 0-100%; and B, 200-500 ml of each of two chicken red blood cells in the step A2, wherein the chicken red blood cells are blood of SPF (specific pathogen free) chicken or cocks, a certain anticoagulant is added during blood collection, the centrifuge tube used in the preparation step is a round-bottom centrifuge tube, the centrifuge tube needs to be turned upside down slowly during centrifugation, the supernatant is discarded until the supernatant is cool and transparent, the specific operation of resuspension is to resuspend the centrifuged cells, active substances and the like by using a proper MACS solution, and the MACS solution containing the cell protective solution comprises 130-480 ml of MACS solution, 3-5 ml of glycerol and 1-3 ml of trehalose.
Examples
The preparation method of the sperm DFI quality control product comprises the following steps:
a1, centrifuging chicken erythrocyte liquid by using a centrifuge, wherein the centrifugation parameters are as follows: centrifuging for 5min at 1000g, setting the temperature at 20 ℃, and keeping the lifting speed at 9;
a2, discarding the supernatant after centrifugation, adding an equal volume of MACS solution, uniformly mixing and resuspending, dividing the chicken red blood cells into two parts and centrifuging;
a3, centrifuging one part of the mixture, removing the supernatant, adding 500ml of MACS solution containing cell protection solution into a centrifuge tube, mixing uniformly, and storing in a refrigerator at 4 ℃ for later use;
a4, opening an oven at 25 ℃ for standby, centrifuging the other part, discarding the supernatant, adding MACS solution with the volume of 500ml into a centrifuge tube for resuspension, mixing uniformly, and adding 30% hydrogen peroxide with the volume of 200ml of suspension. After mixing, putting the mixture into an oven at 25 ℃ for reaction for 1 hour, and paying attention to prevent the centrifugal tube cover from being screwed too tightly;
a5, after the reaction is finished, taking out the centrifuge tube for centrifugation, discarding the supernatant, adding 500ml of MACS solution for cleaning for 3 times, finally resuspending with 500ml of MACS solution containing cell protection solution, and transferring to a new centrifuge tube for later use;
a6, counting two groups of cells by flow cytometry or microscope, can be prepared into cell mixture with DFI value of 0% -100%.
The method has the advantages that after the detection of the step A6, the detection results of two groups of finished products are compared, so that the treatment of the chicken erythrocyte by the hydrogen peroxide to fragment the DNA of the chicken erythrocyte can be obviously compared, the DFI value can be controlled by mixing the chicken erythrocyte with normal cells in a certain proportion, the cells can be stored for a long time at the conventional storage temperature by using the cell protection component, and the DFI value is not influenced by the storage condition.
Although embodiments of the present invention have been shown and described, it is intended that the present invention should not be limited thereto, that the particular features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples, and that modifications, substitutions, variations or the like, which are not inventive and may be made by those skilled in the art without departing from the principle and spirit of the present invention and without departing from the scope of the claims.
Claims (7)
1. A preparation method of quality control products for sperm DNA fragmentation detection comprises the following components used in a sperm DFI quality control product test process: 500ml to 1000ml of chicken erythrocyte liquid, 150ml to 500ml of MACS solution, 100ml to 200ml of hydrogen peroxide with the concentration of 25 percent to 30 percent and 150ml to 500ml of MACS solution containing cell protection solution.
2. The method for preparing a quality control material for sperm DNA fragmentation detection according to claim 1, wherein the method comprises the steps of: the preparation method of the sperm DFI quality control product comprises the following steps:
a1, centrifuging 500-1000 ml of chicken erythrocyte liquid by using a centrifuge for 5-10 min, setting the temperature at 10-40 ℃ and the lifting rate at 5-9 times;
a2, discarding the supernatant after centrifugation, adding 150 ml-500 ml of MACS solution, uniformly mixing and resuspending, dividing the chicken red blood cells into two parts and centrifuging;
a3, centrifuging one part of the mixture, removing the supernatant, adding 150 ml-500 ml of MACS solution containing cell protection solution into a centrifuge tube, mixing uniformly, and storing in a refrigerator at the temperature of 8 ℃ below zero for later use;
a4, opening an oven at 20-25 ℃ for standby, centrifuging the other part, discarding the supernatant, adding 150-500 ml of MACS solution into a centrifuge tube for resuspension, uniformly mixing, adding 100-200 ml of 30% hydrogen peroxide suspension, immediately placing the mixture into the oven at 20-25 ℃ for reaction for 1-2 hours, and paying attention to the fact that the cover of the centrifuge tube is not screwed too tightly;
a5, after the reaction is finished, taking out the centrifuge tube for centrifugation, discarding the supernatant, adding 150 ml-500 ml of MACS solution for cleaning for 3 times, finally resuspending by using 150 ml-500 ml of MACS solution containing cell protection solution, and transferring to a new centrifuge tube for later use;
a6, counting two groups of cells by flow cytometry or microscope, can be prepared into cell mixture with DFI value of 0% -100%.
3. The method for preparing a quality control material for sperm DNA fragmentation detection according to claim 1, wherein the method comprises the steps of: and each of the two chicken red blood cells in the step A2 is 200-500 ml.
4. The method for preparing a quality control material for sperm DNA fragmentation detection according to claim 1, wherein the method comprises the steps of: the centrifuge tube used in the manufacturing step is a round-bottom centrifuge tube, the centrifuge tube needs to be slowly turned upside down when being used for centrifuging, and the supernatant is discarded until the supernatant is cool and transparent.
5. The method for preparing a quality control material for sperm DNA fragmentation detection according to claim 1, wherein the method comprises the steps of: the resuspension is carried out by resuspending the centrifuged cells and active material with the appropriate MACS solution.
6. The method for preparing a quality control material for sperm DNA fragmentation detection according to claim 1, wherein the method comprises the steps of: the components of the MACS solution containing the cell protection solution comprise 130ml to 480ml of MACS solution, 3ml to 5ml of glycerol and 1ml to 3ml of trehalose.
7. The method for preparing a quality control material for sperm DNA fragmentation detection according to claim 1, wherein the method comprises the steps of: the chicken erythrocyte liquid is blood of SPF chicken or cock, and a certain anticoagulant is added during blood collection.
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2021
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| US20080026361A1 (en) * | 2006-06-12 | 2008-01-31 | The Jackson Laboratory | Sperm cryoprotective media |
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