A quality control product for sperm DNA fragmentation detection and its preparation method and application

Abstract

The present invention discloses a quality control product for sperm DNA fragmentation detection, and a preparation method and application thereof. The cell DNA fragmentation method comprises: mixing cells with a DNA fragmentation reagent to obtain DNA-fragmented cells, wherein the DNA fragmentation reagent contains an oxidant and hydrochloric acid. The present invention designs a cell DNA fragmentation method and a preparation method of a quality control product for sperm DFI detection, which can effectively reduce cell loss and reduce costs, and can accurately control the cell DNA fragmentation value and make the quality control product have high stability and uniformity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a quality control product for sperm DNA fragmentation detection and a preparation method and application thereof. Background Art

[0002] At present, the main method for diagnosing male infertility is to detect laboratory indicators such as semen volume, sperm concentration, sperm survival rate, sperm motility and sperm morphology. However, these parameters are affected by the subjective judgment of the experimenter and have a large fluctuation range. These parameters alone can no longer meet clinical needs. In addition, some studies have pointed out that regardless of whether the results of routine semen examinations of male infertility patients are normal, sperm DNA abnormalities may exist.

[0003] Sperm DNA fragmentation index (DFI) refers to the damage to intact sperm DNA and the generation of DNA fragments caused by harmful factors (such as oxidative stress, smoking, high temperature, drugs, etc.) during the formation of sperm. Sperm DFI testing is independent of routine semen examinations. It reflects sperm DNA damage at the molecular level, evaluates male fertility, and can predict the parameters of assisted reproductive clinical outcomes to a certain extent. Many hospitals have carried out sperm DFI testing, among which the sperm chromatin structure analysis (SCSA) method using flow cytometry is the most widely used.

[0004] Currently, the demand for sperm DFI testing is increasing. However, due to the lack of standardized operating procedures and corresponding quality control products, the test results vary and cannot be accurately guaranteed. At present, most reproductive testing laboratories that conduct DFI testing do not conduct internal quality control. Some laboratories often use real sperm samples for internal quality control. The problems are as follows: (1) It cannot meet the needs of inter-laboratory quality control; (2) When using different sperm samples, the uniformity of sperm cells is different, which is more likely to cause intra-batch and inter-batch differences; (3) It requires liquid nitrogen storage and is not easy to preserve; (4) The DNA fragmentation value cannot be accurately controlled; (5) There are many potential ethical and other risks involving human genetic samples, which cannot be used for external quality control; (6) It cannot be promoted on a large scale in clinical practice. In addition, there are reports of using chicken red blood cells to simulate sperm cells as quality control products for sperm DFI detection. For example, CN113432959A discloses a method for preparing a quality control product for sperm DNA fragmentation detection. The chicken red blood cells used are similar in size to human sperm, have a nucleus, are at the end of a stable division cycle, and have a mature production and detection system, which can make up for the difficulties in obtaining raw materials and the shortcomings of being unable to be promoted on a large scale. However, directly using hydrogen peroxide to oxidize the chicken red blood cell DNA to break its DNA chain can easily damage the cell membrane of the chicken red blood cells, resulting in a large loss of chicken red blood cells, resulting in a high preparation cost, which is not conducive to promotion and application, and the resulting DNA fragmentation effect is unstable.

[0005] In summary, how to provide a quality control product for sperm DNA fragmentation detection with low preparation cost and high stability is one of the problems that urgently need to be solved in the field of sperm DNA fragmentation detection. Summary of the invention

[0006] In view of the deficiencies in the prior art and actual needs, the present invention provides a quality control product for sperm DNA fragmentation detection and a preparation method and application thereof, in order to solve many problems such as high cost and difficult application of existing DFI detection quality control products.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for fragmenting cell DNA, the method comprising:

[0009] The cells are mixed with a DNA fragmentation reagent containing an oxidizing agent and hydrochloric acid to obtain DNA-fragmented cells.

[0010] In the present invention, the oxidant in the DNA fragmentation reagent has strong oxidizing properties, can act on phosphodiester bonds and deoxyribose to break the DNA nucleotide chain, and cooperates with hydrochloric acid to efficiently and stably fragment the complete DNA and effectively reduce cell loss.

[0011] Preferably, the oxidizing agent comprises hydrogen peroxide and / or hypochlorous acid.

[0012] In the present invention, selecting a specific oxidant can further improve the fragmentation effect and reduce cell loss.

[0013] Preferably, the concentration of the hydrochloric acid in the DNA fragmentation reagent is 0.08-1.08 mol / L, including but not limited to 0.09 mol / L, 0.1 mol / L, 0.12 mol / L, 0.2 mol / L, 0.3 mol / L, 0.5 mol / L, 0.8 mol / L, 0.9 mol / L, 1 mol / L, 1.02 mol / L, 1.04 mol / L, 1.06 mol / L or 1.07 mol / L.

[0014] Preferably, the mass percentage of the hydrogen peroxide in the DNA fragmentation reagent is 3%-30%, including but not limited to 4%, 5%, 6%, 7%, 8%, 10%, 15%, 20%, 21%, 22%, 25%, 26%, 27%, 28% or 29%.

[0015] Preferably, the concentration of the hypochlorous acid in the DNA fragmentation reagent is 30-300 μg / mL, including but not limited to 31 μg / mL, 32 μg / mL, 33 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, 50 μg / mL, 80 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 220 μg / mL, 250 μg / mL, 260 μg / mL, 280 μg / mL, 290 μg / mL, 291 μg / mL, 292 μg / mL, 295 μg / mL, 296 μg / mL, 298 μg / mL or 299 μg / mL.

[0016] In the present invention, controlling the concentrations of the oxidant and hydrochloric acid in the DNA fragmentation reagent can further improve the fragmentation effect and reduce cell loss.

[0017] Preferably, the DNA fragmentation reagent further contains a surfactant and / or an inorganic salt.

[0018] Preferably, the surfactant comprises any one of polyethylene glycol octylphenyl ether, polysorbate-20, polysorbate-80, dodecyl maltoside, digitonin or ethylphenyl polyethylene glycol, or a combination of at least two thereof.

[0019] Preferably, the inorganic salt includes any one of sodium chloride, potassium chloride, magnesium chloride, ferrous chloride, ferric chloride, calcium chloride, potassium dihydrogen phosphate or sodium dihydrogen phosphate, or a combination of at least two thereof.

[0020] Preferably, the step of pre-fixing the cells is also included before the mixing.

[0021] Preferably, the pre-fixing step comprises:

[0022] Mix the cells with the cell fixative.

[0023] In the present invention, a clever design is used to perform a fixation treatment on cells before fragmentation, which can effectively reduce cell loss and prolong the cell preservation time.

[0024] Preferably, the cell fixative solution contains a cell fixative.

[0025] Preferably, the cell fixative comprises formaldehyde and / or glutaraldehyde.

[0026] In the present invention, the use of a specific fixative can further reduce cell loss and prolong cell preservation time.

[0027] Preferably, the mass percentage of formaldehyde in the cell fixative is 3%-10%, including but not limited to 4%, 5%, 6%, 7%, 8% or 9%.

[0028] Preferably, the mass percentage of glutaraldehyde in the cell fixative is 2.5%-5%, including but not limited to 2.6%, 2.7%, 2.8%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 4.6%, 4.8% or 4.9%.

[0029] Preferably, the cell fixative further contains a surfactant and / or an inorganic salt.

[0030] Preferably, the surfactant comprises any one of polyethylene glycol octylphenyl ether, polysorbate-20, polysorbate-80, dodecyl maltoside, digitonin or ethylphenyl polyethylene glycol, or a combination of at least two thereof.

[0031] Preferably, the inorganic salt includes any one of sodium chloride, potassium chloride, magnesium chloride, ferrous chloride, ferric chloride, calcium chloride, potassium dihydrogen phosphate or sodium dihydrogen phosphate, or a combination of at least two thereof.

[0032] Preferably, the method further comprises the step of mixing the DNA-fragmented cells with a cell protection solution.

[0033] Preferably, the cell protection solution contains an antibacterial agent, a chelating agent and an amino acid.

[0034] In the present invention, mixing DNA-fragmented cells with a cell protection solution of a specific component can effectively maintain the stability of the fragmented DNA, which is conducive to long-term storage. In addition, the use of the cell protection solution can allow the cells to be stored at a conventional storage temperature for a certain period of time, and still maintain good stability of the fragmented DNA.

[0035] In the present invention, the use of amino acids can effectively quench free aldehyde groups and avoid their influence on DFI detection and the like.

[0036] Preferably, the bacteriostatic agent comprises imidazolidinyl urea.

[0037] Preferably, the chelating agent includes any one of disodium ethylenediaminetetraacetate, disodium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, tripotassium ethylenediaminetetraacetate, tetrasodium ethylenediaminetetraacetate or disodium nitrilotriacetate, or a combination of at least two thereof.

[0038] Preferably, the amino acids include any one of glycine, lysine, arginine or aspartic acid, or a combination of at least two of them.

[0039] Preferably, the cell protection solution further contains phosphate buffer, trehalose, bovine serum albumin, Tween and glycerol.

[0040] As a preferred technical solution, the cell DNA fragmentation method comprises:

[0041] The cells are mixed with a cell fixative to obtain fixed cells, and the fixed cells are mixed with a DNA fragmentation reagent to obtain DNA-fragmented cells. The cell fixative contains a cell fixative, and the DNA fragmentation reagent contains an oxidant and hydrochloric acid.

[0042] In a second aspect, the present invention provides a method for preparing a quality control product for sperm fragmentation detection, the method comprising:

[0043] The raw material cells are fragmented using the cell DNA fragmentation method described in the first aspect to obtain DNA-fragmented cells, and the DNA-fragmented cells are mixed with the raw material cells in proportion to obtain the quality control product for sperm fragmentation detection.

[0044] The present invention ingeniously designs a cell DNA fragmentation method and prepares a quality control product for sperm fragmentation detection, with less cell loss, which can reduce the cost of quality control product preparation; the prepared cell fragmentation effect is stable, the cell DNA fragmentation value can be accurately controlled, the uniformity and stability of the cells can be guaranteed, and large-scale, industrialized production can be carried out; the quality control requirements of sperm DFI detection can be met, and the DFI detection items can be quality controlled between laboratories; it is easy to store and can be stored for a long time under conventional storage temperature conditions; analysis can be performed using the counting software provided by the flow cytometer, which greatly facilitates clinical and scientific research workers; the quality control is stable and convenient for large-scale clinical promotion.

[0045] Preferably, the raw material cells include chicken red blood cells.

[0046] In the present invention, the chicken red blood cells can be taken from healthy male roosters or SPF chickens.

[0047] It can be understood that the proportion of cells with DNA fragmentation in the quality control product for sperm fragmentation detection can be set according to needs.

[0048] Preferably, the percentage of the number of cells with DNA fragmentation in the sperm fragmentation detection quality control product is 0% to 100%, for example, it can be 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 15%, 20%, 50%, 70%, 80%, 90%, 91%, 92%, 93%, 96%, 98% or 99%.

[0049] In a third aspect, the present invention provides a quality control product for sperm fragmentation detection, wherein the quality control product for sperm fragmentation detection is prepared by the method for preparing a quality control product for sperm fragmentation detection described in the second aspect.

[0050] The quality control product for sperm fragmentation detection prepared by the present invention has high uniformity and stability, can be mass-produced and industrialized, meets the quality control requirements of sperm DFI detection, and can perform quality control on DFI detection items between laboratories, is easy to store, and can be stored for a long time under conventional storage temperature conditions, can be analyzed using the counting software provided by the flow cytometer, and is greatly convenient for clinical and scientific researchers, has stable quality control, and is convenient for large-scale clinical promotion.

[0051] In a fourth aspect, the use of the quality control product for sperm fragmentation detection described in the third aspect in the preparation of sperm fragmentation detection products.

[0052] In a fifth aspect, the present invention provides a sperm fragmentation detection kit, the kit comprising the quality control product for sperm fragmentation detection described in the third aspect;

[0053] Preferably, the kit further comprises a flow cytometry detection reagent.

[0054] It can be understood that the quality control product for sperm fragmentation detection of the present invention can be used in combination with the DFI detection kit to perform quality control on the DFI detection items, ensure the reliability of the reagent performance and the operation method, and solve the problem that conventional sperm DFI quality control products are high in cost and difficult to promote on a large scale.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] (1) The cell DNA fragmentation method and the preparation method of the quality control product for sperm DFI detection designed by the present invention can effectively reduce cell loss and reduce costs, and at the same time can accurately control the cell DNA fragmentation value and make the quality control product have high stability and uniformity;

[0057] (2) The quality control product for sperm fragmentation detection prepared by the present invention has high uniformity and stability, and can be mass-produced and industrialized; it meets the quality control requirements of sperm DFI detection, and can perform quality control on DFI detection items between laboratories; it is easy to store and can be stored for a long time under conventional storage temperature conditions; it can be analyzed using the counting software provided by the flow cytometer, which greatly facilitates clinical and scientific research workers; the quality control is stable and convenient for large-scale clinical promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Flow chart for preparing quality control products for sperm fragmentation detection;

[0059] Figure 2 The peak graph shows a mixture of normal cells and fragmented cells with a DFI value of 5%;

[0060] Figure 3 The peak graph shows a mixture of normal cells and fragmented cells with a DFI value of 15%;

[0061] Figure 4 The peak graph shows a mixture of normal cells and fragmented cells with a DFI value of 30%;

[0062] Figure 5 The peak graph shows a mixture of normal cells and fragmented cells with a DFI value of 50%;

[0063] Figure 6 The peak graph shows a mixture of normal cells and fragmented cells with a DFI value of 70%;

[0064] Figure 7 The double fluorescence scatter plot is a mixture of normal cells and fragmented cells with a DFI value of 5%;

[0065] Figure 8 The double fluorescence scatter plot is a mixture of normal cells and fragmented cells with a DFI value of 15%;

[0066] Fig. 9 The double fluorescence scatter plot is a mixture of normal cells and fragmented cells with a DFI value of 30%;

[0067] Fig.10 The double fluorescence scatter plot is a mixture of normal cells and fragmented cells with a DFI value of 50%;

[0068] Fig.11 The double fluorescence scatter plot shows that normal cells and fragmented cells are mixed into a DFI value of 70%. DETAILED DESCRIPTION

[0069] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0070] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0071] In the specific embodiment of the present invention, chicken red blood cells are used as an example to verify the method for preparing a quality control product for cell DNA fragmentation and sperm DFI detection of the present invention. The schematic diagram of the process is shown in FIG. Figure 1As shown, the reagents used can be selected as follows (wherein the % involved are all mass percentages): cell fixative, DNA fragmentation reagent, cell protection solution and 1% to 20% chicken red blood cells. The cell fixative is prepared according to the following components and concentration ranges: 3% to 10% formaldehyde or 2.5% to 5% glutaraldehyde, 30% to 40% glycerol and 0.6% to 1% sodium chloride; the DNA fragmentation reagent is prepared according to the following components and concentration ranges: 3% to 30% hydrogen peroxide or 30-300μg / mL hypochlorous acid, 0.08-1.08mol / L hydrochloric acid, 0.15-0.65mol / L sodium chloride and 0.1% to 1% Triton-X 100; the cell protection solution is prepared according to the following components and concentration ranges: 1% to 5% imidazolidinyl urea, 0.1% to 1% EDTA-2Na (disodium ethylenediaminetetraacetic acid), phosphate buffer, 4.0-10g / L trehalose, 0.1% to 3% glycine, 1.0 to 2.5g / L bovine serum albumin, 0.01% to 0.05% Tween-20, 30% to 40% glycerol; the pH is adjusted to 7.4. Chicken red blood cells can be taken from healthy male adult roosters or SPF chickens, and heparin anticoagulated blood is collected from the vein under the wings under aseptic operation. After centrifugation, chicken red blood cell sediment is obtained, the white blood cell layer and supernatant are discarded, and the supernatant is washed with an equal amount of phosphate solution until the supernatant is transparent, and finally the chicken red blood cells are prepared with Aldrich solution.

[0072] Example 1

[0073] This embodiment provides a method for preparing a quality control product for sperm fragmentation detection.

[0074] 1. Prepare the reagents:

[0075] The cell fixative was prepared according to the following components and concentrations: 3.5% formaldehyde, 40% glycerol, and 0.6% sodium chloride, and the remaining components were supplemented with phosphate buffer;

[0076] The DNA fragmentation reagent formula was prepared according to the following components and concentrations: 10% hydrogen peroxide, 0.08 mol / L hydrochloric acid, 0.65 mol / L sodium chloride, and 1% Triton-X 100, and the remaining components were supplemented with phosphate buffer;

[0077] The cell protection solution was prepared according to the following components and concentrations: 1% imidazolidinyl urea, 0.1% EDTA-2Na (disodium ethylenediaminetetraacetic acid), phosphate buffer, 6 g / L trehalose, 0.3% glycine, 4.0 g / L glycerol, 1.0 g / L bovine serum albumin, 0.05% Tween-20, 40% glycerol; the pH was adjusted to 7.4 and sterilized by filtration through a 0.22 μm filter membrane.

[0078] 2. Fixation of sperm-like cells:

[0079] A1. Take 200 mL of 1% chicken red blood cells and centrifuge them. The centrifuge setting parameters are 5000 rpm, 5 min, and 25°C.

[0080] A2. After centrifugation, discard the supernatant, add 200 mL of phosphate buffer to resuspend the cells, mix well and centrifuge, repeat once;

[0081] A3. After centrifugation, discard the supernatant and add 200 mL of cell fixation reagent, resuspend and mix well, and place in a 4°C refrigerator for 24 hours;

[0082] A4. Take out 200 mL of fixed chicken red blood cells and divide them into two equal parts. Centrifuge and discard the supernatant. Add 200 mL of phosphate buffer to resuspend and mix. Centrifuge and discard the supernatant. Repeat once. Count on a flow cytometer and adjust the chicken red blood cell concentration to 1×10 8 / mL; after finishing, mix well, divide into 2 tubes and centrifuge;

[0083] A5. After centrifugation and discarding the supernatant, add one portion of the chicken red blood cells to 100 mL of cell protection solution and place it in a 4°C refrigerator for later use.

[0084] 3. Fragmentation of Cellular DNA

[0085] A6. Open the incubator in advance and set the temperature of the incubator to 25°C; add another portion of chicken red blood cells to 600 mL of cell fragmentation reagent, resuspend the cells and mix well, and place in the incubator to react for 2 hours.

[0086] A7. After the reaction is completed, centrifuge and discard the supernatant, then add 100 mL PBS buffer and wash twice.

[0087] 4. Preparation and Storage of DFI Quality Control Products

[0088] A8, after the chicken red blood cells obtained in A7 are centrifuged, add 100 mL of cell protection solution and mix well for later use, and take out the chicken red blood cells obtained in A5;

[0089] A9. Count the two chicken red blood cells accurately under a microscope and adjust the concentration of the two cells to be consistent (no less than 20×10 6 / mL), and a cell mixture with a DFI of 0 to 100% is prepared according to the count, which is a quality control product for sperm fragmentation detection. For example, Figure 2 to Figure 6 The peak graphs of normal cells and fragmented cells mixed with different DFI values ​​(DFI values ​​are 5%, 15%, 30%, 50%, and 70%) are shown respectively. Figure 7 to Figure 11Dual fluorescence scatter plots of normal cells and fragmented cells mixed with different DFI values ​​(DFI values ​​are 5%, 15%, 30%, 50%, and 70%) are shown respectively. The cell population on the left side of each graph is normal cells, i.e., a cell population with good DNA integrity; the cell population on the right side of the graph is fragmented cells, i.e., a cell population with poor DNA integrity.

[0090] Example 2

[0091] This embodiment provides a method for preparing a quality control product for sperm fragmentation detection.

[0092] 1. Prepare the reagents:

[0093] The cell fixative was prepared according to the following components and concentrations: 3% formaldehyde, 30% glycerol, and 1% sodium chloride, and the remaining components were supplemented with phosphate buffer;

[0094] The DNA fragmentation reagent formula was prepared according to the following components and concentrations: 30% hydrogen peroxide, 1.08 mol / L hydrochloric acid, 0.15 mol / L sodium chloride, and 0.1% Triton-X 100, and the remaining components were supplemented with phosphate buffer;

[0095] The cell protection solution was prepared according to the following components and concentrations: 5% imidazolidinyl urea, 0.1% EDTA-2Na (disodium ethylenediaminetetraacetic acid), phosphate buffer, 6 g / L trehalose, 1% glycine, 10.0 g / L glycerol, 1.0 g / L bovine serum albumin, 0.05% Tween-20, and 30% glycerol; the pH was adjusted to 7.4 and sterilized by filtration through a 0.22 μm filter membrane.

[0096] 2. Fixation of sperm-like cells:

[0097] A1. Take 200 mL of 1% chicken red blood cells and centrifuge them. The centrifuge setting parameters are 5000 rpm, 5 min, and 25°C.

[0098] A2. After centrifugation, discard the supernatant, add 200 mL of phosphate buffer to resuspend the cells, mix well and centrifuge, repeat once;

[0099] A3. After centrifugation, discard the supernatant and add 200 mL of cell fixation reagent, resuspend and mix well, and place in a 4°C refrigerator for 24 hours;

[0100] A4. Take out 200 mL of fixed chicken red blood cells and divide them into two equal parts. Centrifuge and discard the supernatant. Add 200 mL of phosphate buffer to resuspend and mix. Centrifuge and discard the supernatant. Repeat once. Count on a flow cytometer and adjust the chicken red blood cell concentration to 1×10 8 / mL; after finishing, mix well, divide into 2 tubes and centrifuge;

[0101] A5. After centrifugation and discarding the supernatant, add one portion of the chicken red blood cells to 100 mL of cell protection solution and place it in a 4°C refrigerator for later use.

[0102] 3. Fragmentation of Cellular DNA

[0103] A6. Open the incubator in advance and set the temperature of the incubator to 25°C; add another portion of chicken red blood cells to 600 mL of cell fragmentation reagent, resuspend the cells and mix well, and place in the incubator to react for 2 hours.

[0104] A7. After the reaction is completed, centrifuge and discard the supernatant, then add 100 mL PBS buffer and wash twice.

[0105] 4. Preparation and Storage of DFI Quality Control Products

[0106] A8, after the chicken red blood cells obtained in A7 are centrifuged, add 100 mL of cell protection solution and mix well for later use, and take out the chicken red blood cells obtained in A5;

[0107] A9. Count the two chicken red blood cells accurately under a microscope and adjust the concentration of the two cells to be consistent (no less than 20×10 6 / mL), and a cell mixture with a DFI of 0-100% is prepared according to the count, which is the quality control product for sperm fragmentation detection.

[0108] Example 3

[0109] This embodiment provides a method for preparing a quality control product for sperm fragmentation detection.

[0110] 1. Prepare the reagents:

[0111] The cell fixative was prepared according to the following components and concentrations: 10% formaldehyde, 30% glycerol, and 1% sodium chloride, and the remaining components were supplemented with phosphate buffer;

[0112] The DNA fragmentation reagent formula was prepared according to the following components and concentrations: 3% hydrogen peroxide, 0.09 mol / L hydrochloric acid, 0.15 mol / L sodium chloride and 0.1% Triton-X 100, and the remaining components were supplemented with phosphate buffer;

[0113] The cell protection solution was prepared according to the following components and concentrations: 5% imidazolidinyl urea, 0.1% EDTA-2Na (disodium ethylenediaminetetraacetic acid), phosphate buffer, 6 g / L trehalose, 1% glycine, 10.0 g / L glycerol, 1.0 g / L bovine serum albumin, 0.05% Tween-20, and 30% glycerol; the pH was adjusted to 7.4 and sterilized by filtration through a 0.22 μm filter membrane.

[0114] 2. Fixation of sperm-like cells:

[0115] A1. Take 200 mL of 1% chicken red blood cells and centrifuge them. The centrifuge setting parameters are 5000 rpm, 5 min, and 25°C.

[0116] A2. After centrifugation, discard the supernatant, add 200 mL of phosphate buffer to resuspend the cells, mix well and centrifuge, repeat once;

[0117] A3. After centrifugation, discard the supernatant and add 200 mL of cell fixation reagent, resuspend and mix well, and place in a 4°C refrigerator for 24 hours;

[0118] A4. Take out 200 mL of fixed chicken red blood cells and divide them into two equal parts. Centrifuge and discard the supernatant. Add 200 mL of phosphate buffer to resuspend and mix. Centrifuge and discard the supernatant. Repeat once. Count on a flow cytometer and adjust the chicken red blood cell concentration to 1×10 8 / mL; after finishing, mix well, divide into 2 tubes and centrifuge;

[0119] A5. After centrifugation and discarding the supernatant, add one portion of the chicken red blood cells to 100 mL of cell protection solution and place it in a 4°C refrigerator for later use.

[0120] 3. Fragmentation of Cellular DNA

[0121] A6. Open the incubator in advance and set the temperature of the incubator to 25°C; add another portion of chicken red blood cells to 600 mL of cell fragmentation reagent, resuspend the cells and mix well, and place in the incubator to react for 2 hours.

[0122] A7. After the reaction is completed, centrifuge and discard the supernatant, then add 100 mL PBS buffer and wash twice.

[0123] 4. Preparation and Storage of DFI Quality Control Products

[0124] A8, after the chicken red blood cells obtained in A7 are centrifuged, add 100 mL of cell protection solution and mix well for later use, and take out the chicken red blood cells obtained in A5;

[0125] A9. Count the two chicken red blood cells accurately under a microscope and adjust the concentration of the two cells to be consistent (no less than 20×10 6 / mL), and a cell mixture with a DFI of 0-100% is prepared according to the count, which is the quality control product for sperm fragmentation detection.

[0126] Example 4

[0127] This embodiment provides a method for preparing a quality control product for sperm fragmentation detection.

[0128] 1. Prepare the reagents:

[0129] The DNA fragmentation reagent formula was prepared according to the following components and concentrations: 10% hydrogen peroxide, 0.08 mol / L hydrochloric acid;

[0130] The cell protection solution was prepared according to the following components and concentrations: 1% imidazolidinyl urea, 0.1% EDTA-2Na (disodium ethylenediaminetetraacetic acid), phosphate buffer, 6 g / L trehalose, 0.3% glycine, 4.0 g / L glycerol, 1.0 g / L bovine serum albumin, 0.05% Tween-20, 40% glycerol; the pH was adjusted to 7.4 and sterilized by filtration through a 0.22 μm filter membrane.

[0131] 2. Fragmentation of Cellular DNA

[0132] A1. Take 200 mL of 1% chicken red blood cells and centrifuge them. The centrifuge setting parameters are 5000 rpm, 5 min, and 25°C.

[0133] A2. After centrifugation, discard the supernatant, add 200 mL of phosphate buffer to resuspend the cells, mix well and centrifuge, repeat once; adjust the chicken red blood cell concentration to 1×10 8 / mL; after finishing, mix well, divide into 2 tubes and centrifuge;

[0134] A3. After centrifugation and discarding the supernatant, add one portion of the chicken red blood cells to 100 mL of cell protection solution and place it in a 4°C refrigerator for later use;

[0135] A4. Open the incubator in advance and set the temperature of the incubator to 25°C. Add another portion of chicken red blood cells to 600 mL of cell fragmentation reagent to resuspend the cells and mix well. Place the cells in the incubator to react for 2 hours.

[0136] A5. After the reaction is completed, centrifuge and discard the supernatant, then add 100 mL PBS buffer and wash twice.

[0137] 3. Preparation and Storage of DFI Quality Control Products

[0138] A6, after the chicken red blood cells obtained in A5 are centrifuged, add 100 mL of cell protection solution and mix well for later use, and take out the chicken red blood cells obtained in A3;

[0139] A7. Count the two chicken red blood cells accurately under a microscope and adjust the concentration of the two cells to be consistent (no less than 20×10 6 / mL), and a cell mixture with a DFI of 0-100% is prepared according to the count, which is the quality control product for sperm fragmentation detection.

[0140] Example 5

[0141] This embodiment provides a method for preparing a quality control product for sperm fragmentation detection.

[0142] Compared with Example 1, the only difference is that the cell fixative is prepared according to the following components and concentrations: 3.5% glutaraldehyde, 40% glycerol and 0.6% sodium chloride, and the rest is the same as Example 1.

[0143] Example 6

[0144] This embodiment provides a method for preparing a quality control product for sperm fragmentation detection.

[0145] Compared with Example 1, the only difference is that the cell fixative is prepared according to the following components and concentrations: 3.5% ethanol, 40% glycerol and 0.6% sodium chloride, and the rest is the same as Example 1.

[0146] Example 7

[0147] This embodiment provides a method for preparing a quality control product for sperm fragmentation detection.

[0148] Compared with Example 1, the only difference is that the DNA fragmentation reagent formula is prepared according to the following components and concentrations: 10% hypochlorous acid, 0.08 mol / L hydrochloric acid, 0.65 mol / L sodium chloride and 1% Triton-X 100, and the rest is the same as Example 1.

[0149] Example 8

[0150] This embodiment provides a method for preparing a quality control product for sperm fragmentation detection.

[0151] Compared with Example 1, the only difference is that the hydrogen peroxide in the DNA fragmentation reagent is 1%, and the rest is the same as Example 1.

[0152] Example 9

[0153] This embodiment provides a method for preparing a quality control product for sperm fragmentation detection.

[0154] Compared with Example 1, the only difference is that the hydrogen peroxide in the DNA fragmentation reagent is 40%, and the rest is the same as Example 1.

[0155] Example 10

[0156] This embodiment provides a method for preparing a quality control product for sperm fragmentation detection.

[0157] Compared with Example 1, the only difference is that the hydrochloric acid in the DNA fragmentation reagent is 0.04 mol / L, and the rest is the same as Example 1.

[0158] Embodiment 11

[0159] This embodiment provides a method for preparing a quality control product for sperm fragmentation detection.

[0160] Compared with Example 1, the only difference is that the hydrochloric acid in the DNA fragmentation reagent is 2 mol / L, and the rest is the same as Example 1.

[0161] Comparative Example 1

[0162] This comparative example provides a method for preparing a quality control product for sperm fragmentation detection.

[0163] Compared with Example 1, the only difference is that the hydrochloric acid in the DNA fragmentation reagent is increased in proportion to hydrogen peroxide, and the rest is the same as Example 1.

[0164] Test Example 1

[0165] This test example analyzes the cell loss and DNA fragmentation effects of the preparation methods of Examples 1-11 and Comparative Example 1.

[0166] After treating the cells with or without a cell fixative, the cells were counted and recorded on a flow cytometer; after the cell concentration was known, the cells were fragmented, and the number of cells after fragmentation was recorded; the number of fragmented cells obtained in each embodiment was compared with the loss rate of the number of cells before or after fixation or non-fixation as shown in Table 1. The present invention can effectively fragment and reduce cell loss, with the lowest loss rate being 9%. By comparing the results of Example 1 with those of Example 4, it can be seen that using hydrogen peroxide and hydrochloric acid as fragmentation agents can effectively fragment, but without cell fixation, the cell loss rate increases, indicating that the present invention can further reduce the cell loss rate by performing cell fixation; By comparing the results of Example 1 with those of Example 4, it can be seen that using hydrogen peroxide and hydrochloric acid as fragmentation agents can effectively perform fragmentation, but without performing cell fixation, the cell loss rate increases, indicating that the present invention can further reduce the cell loss rate by performing cell fixation; Comparison of the results of Examples 5 and 6 shows that the use of a fixing solution containing formaldehyde can further reduce the loss rate; comparison of the results of Example 1 with Example 7 shows that the use of hydrogen peroxide as an oxidant in a DNA fragmentation reagent can further reduce the loss rate; comparison of the results of Example 1 with Comparative Example 1 shows that it is difficult to fragment using only hydrogen peroxide, and the cell loss rate increases; comparison of the results of Example 1 with Examples 8 and 9 shows that the present invention controls the amount of hydrogen peroxide, which can achieve complete fragmentation and ensure a low loss rate; comparison of the results of Example 1 with Examples 10 and 11 shows that the present invention controls the amount of hydrochloric acid, which can achieve complete fragmentation and ensure a low loss rate.

[0167] Table 1

[0168]

[0169] Test Example 2

[0170] This test example tests the stability of the quality control products for sperm fragmentation detection prepared in Examples 1-11 and Comparative Example 1.

[0171] The quality control products prepared in Examples 1-11 and Comparative Example 1 were stored at 4°C, and the fragmentation rates of the quality control products were detected using the DFI flow cytometry kit at 0 days, 7 days, 15 days, 30 days, 3 months, and 6 months, respectively. The fragmentation rate of the quality control product of each embodiment was recorded, and the relative deviation and the concentration loss of the quality control product were calculated. The results are shown in Table 2. The quality control product prepared by the present invention can be stored for a long time. The relative deviation of the fragmentation rate after 6 months of storage is not higher than 50%, and the lowest is only 5%. Comparing the results of Example 1 with those of Example 4, it can be seen that the cell fixation of the present invention can further improve the stability of the quality control product; comparing the results of Example 1 with those of Examples 5 and 6, it can be seen that the use of a fixative containing formaldehyde can further improve the stability; comparing the results of Example 1 with those of Example 7, it can be seen that using hydrogen peroxide as an oxidant in a DNA fragmentation reagent can further improve the stability; comparing the results of Example 1 with those of Comparative Example 1, it can be seen that the stability of the quality control product is reduced by using only hydrogen peroxide; comparing the results of Example 1 with those of Examples 8 and 9, it can be seen that the present invention controls the amount of hydrogen peroxide to further improve the stability of the quality control product; comparing the results of Example 1 with those of Examples 10 and 11, it can be seen that the present invention controls the amount of hydrochloric acid to further improve the stability of the quality control product.

[0172] Table 2

[0173]

[0174] Test Example 3

[0175] This test example tests the uniformity of the quality control products for sperm DNA fragmentation detection with a sperm fragmentation rate of 40% prepared in Examples 1-11 and Comparative Example 1.

[0176] After the quality control product was stored for 1 month, 10 bottles of the quality control product of the same batch when each example was made were taken, and their DNA fragmentation values ​​were detected, and the mean, standard deviation and coefficient of variation (CV) of each example were calculated to verify the uniformity of different packaged products of the same batch; in addition, 1 bottle was randomly taken out from each of the 10 bottles of each of the above examples, and the same number of continuous sampling tests were performed, and the mean, standard deviation and coefficient of variation (CV) of the test results were calculated to verify the uniformity in the same packaged product.

[0177] The results are shown in Table 3 (wherein the first row of each example is the test data of 10 quality control products from the same batch, and the second row is the data of 10 consecutive sampling tests of the same quality control product). The quality control products prepared by the present invention have good uniformity, and the uniformity in the same packaged product is good, with the average CV value within 3%, and the repeatability CV value between bottles is not higher than 11.4%. The uniformity of the quality control products tested in Example 1 according to the above technical guidelines is better than that of other examples.

[0178] Table 3

[0179]

[0180] In summary, the cell DNA fragmentation method and the preparation method of the quality control product for sperm DFI detection designed by the present invention can effectively reduce cell loss and reduce costs, and at the same time can accurately control the cell DNA fragmentation value and make the quality control product have high stability and uniformity; the quality control product for sperm fragmentation detection prepared by the present invention has high uniformity and stability, and can be mass-produced and industrialized; it meets the quality control requirements of sperm DFI detection, and the DFI detection items can be quality controlled between laboratories; it is easy to store and can be stored for a long time under conventional storage temperature conditions; it can be analyzed using the counting software provided by the flow cytometer, which greatly facilitates clinical and scientific research workers; the quality control is stable and convenient for large-scale clinical promotion.

[0181] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for fragmenting cell DNA, characterized in that: The cell DNA fragmentation method comprises: mixing the cells with a DNA fragmentation reagent to obtain DNA-fragmented cells; mixing the DNA-fragmented cells with a cell protection solution; The DNA fragmentation reagent contains an oxidant and hydrochloric acid; The oxidant includes hydrogen peroxide; The mass percentage of the hydrogen peroxide is 3%-22%; the concentration of the hydrochloric acid is 0.08-1.08 mol / L; The cell protection solution contains antibacterial agent, chelating agent and amino acid.

2. The method for cell DNA fragmentation according to claim 1, characterized in that: The DNA fragmentation reagent further contains a surfactant and / or an inorganic salt.

3. The method for cell DNA fragmentation according to claim 2, characterized in that: The surfactant includes any one of polyethylene glycol octylphenyl ether, polysorbate-20, polysorbate-80, dodecyl maltoside, digitonin or ethylphenyl polyethylene glycol, or a combination of at least two thereof.

4. The method for cell DNA fragmentation according to claim 2, characterized in that: The inorganic salt includes any one of sodium chloride, potassium chloride, magnesium chloride, ferrous chloride, ferric chloride, calcium chloride, potassium dihydrogen phosphate or sodium dihydrogen phosphate, or a combination of at least two thereof.

5. The method for cell DNA fragmentation according to claim 1, characterized in that: The method also includes a step of pre-fixing the cells before the mixing.

6. The method for cell DNA fragmentation according to claim 5, characterized in that: The pre-fixing step comprises: Mix the cells with the cell fixative.

7. The method for cell DNA fragmentation according to claim 6, characterized in that: The cell fixative solution contains a cell fixative.

8. The method for cell DNA fragmentation according to claim 7, characterized in that: The cell fixative includes formaldehyde and / or glutaraldehyde.

9. The method for fragmenting cell DNA according to claim 6, characterized in that: The cell fixative further contains a surfactant and / or an inorganic salt.

10. The method for fragmenting cell DNA according to claim 9, characterized in that: The surfactant includes any one of polyethylene glycol octylphenyl ether, polysorbate-20, polysorbate-80, dodecyl maltoside, digitonin or ethylphenyl polyethylene glycol, or a combination of at least two thereof.

11. The method for fragmenting cell DNA according to claim 9, characterized in that: The inorganic salt includes any one of sodium chloride, potassium chloride, magnesium chloride, ferrous chloride, ferric chloride, calcium chloride, potassium dihydrogen phosphate or sodium dihydrogen phosphate, or a combination of at least two thereof.

12. The method for fragmenting cell DNA according to claim 1, characterized in that: The bacteriostatic agent includes imidazolidinyl urea.

13. The method for fragmenting cell DNA according to claim 1, characterized in that: The chelating agent includes any one of disodium ethylenediaminetetraacetate, disodium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, tripotassium ethylenediaminetetraacetate, tetrasodium ethylenediaminetetraacetate or disodium nitrilotriacetate, or a combination of at least two thereof.

14. The method for fragmenting cell DNA according to claim 1, characterized in that: The amino acids include any one of glycine, lysine, arginine or aspartic acid, or a combination of at least two of them.

15. The method for fragmenting cell DNA according to claim 1, characterized in that: The cell protection solution also contains phosphate buffer, trehalose, bovine serum albumin, Tween and glycerol.

16. The method for fragmenting cell DNA according to claim 1, characterized in that: The method comprises: mixing the cells with a cell fixative to obtain fixed cells, and mixing the fixed cells with a DNA fragmentation reagent to obtain DNA-fragmented cells; The cell fixative solution contains a cell fixative; The DNA fragmentation reagent contains an oxidizing agent and hydrochloric acid.

17. A method for preparing a quality control product for sperm fragmentation detection, characterized in that: The method comprises: The raw material cells are fragmented using the cell DNA fragmentation method described in any one of claims 1 to 16 to obtain DNA-fragmented cells, and the DNA-fragmented cells are mixed with the raw material cells in proportion to obtain the sperm fragmentation detection quality control product.

18. The method for preparing a quality control product for sperm fragmentation detection according to claim 17, characterized in that: The raw material cells include chicken red blood cells.

19. The method for preparing a quality control product for sperm fragmentation detection according to claim 17, characterized in that: The percentage of the number of cells with DNA fragmentation in the quality control product for sperm fragmentation detection is 0% to 100%.

20. A quality control product for sperm fragmentation detection, characterized in that: The quality control product for sperm fragmentation detection is prepared by the method for preparing a quality control product for sperm fragmentation detection according to any one of claims 17 to 19.

21. Use of the quality control product for sperm fragmentation detection according to claim 20 in the preparation of sperm fragmentation detection products.

22. A sperm fragmentation detection kit, characterized in that: The kit includes the quality control product for sperm fragmentation detection as described in claim 20.

23. The sperm fragmentation detection kit according to claim 22, characterized in that: The kit also includes flow cytometry detection reagents.

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