CN111307696A

CN111307696A - Method and kit for detecting sperm DNA fragmentation rate

Info

Publication number: CN111307696A
Application number: CN202010196442.3A
Authority: CN
Inventors: 曾桥; 胡西陵; 陈继勇
Original assignee: Zhejiang Cellpro Biotech Co ltd
Current assignee: Zhejiang Cellpro Biotech Co ltd
Priority date: 2020-03-19
Filing date: 2020-03-19
Publication date: 2020-06-19

Abstract

Methods and kits for detecting sperm DNA fragmentation rates are provided. The method and the kit provided by the application are based on flow cytometry, RNA in a sperm sample is digested by utilizing RNA enzyme, so that mixed signals brought by the sperm RNA are eliminated, the detection result is more accurate, stable and reliable, the repeatability is good, and the clinical popularization is easy.

Description

Method and kit for detecting sperm DNA fragmentation rate

Technical Field

The application belongs to the field of in-vitro diagnostic reagents, and particularly relates to a method and a kit for detecting sperm DNA fragmentation rate.

Background

DNA is a long chain polymer composed of four deoxynucleotides in units of: adenine deoxynucleotide (dAMP), thymine deoxynucleotide (dTMP), cytosine deoxynucleotide (dCMP), guanine deoxynucleotide (dGMP). Deoxyribose (pentose) and phosphate molecules are connected through ester bonds to form a DNA long-chain framework which is arranged on the outer side, and four bases are arranged on the inner side and follow the base complementary pairing principle. Each sugar molecule is linked to one of four bases, which are arranged along a long DNA chain to form a sequence that can constitute the genetic code directing the synthesis of proteins.

The primary function of sperm is to retain and transmit genetic information of parents. Each normal sperm contains 23 chromosomes, wherein the DNA content is 3.32 pg/sperm, the RNA content is 0.2-0.3 pg/sperm, and the RNA content reaches 6.02-9.04% of the DNA content of the sperm.

The DNA is present in the sperm and is encapsulated by protamine in a dense structure. Since DNA stores the genetic information on which organisms live and reproduce, maintaining the integrity of DNA molecules is crucial to cells. In contrast to RNA and proteins which can be synthesized in large amounts in cells, there is usually only one DNA in a prokaryotic cell and only one pair of identical DNAs in a eukaryotic diploid cell, and if damage to DNA or alteration of genetic information cannot be corrected, the function or survival of somatic cells may be affected, and the progeny of germ cells may be affected. Under normal conditions, DNA molecules are complete double-stranded structures, DNA damage can be caused in vivo due to various reasons, and the damaged DNA can have great influence on the multiplication of biological individuals, so that the fragmentation degree of sperm DNA reflects the integrity of sperm genetic materials, and the method is of great significance for sperm DNA integrity detection.

The methods commonly used for sperm DNA integrity testing are:

(1) sperm chromatin structure analysis (scea): the method can carry out quantitative detection, has high sensitivity, convenient operation and short detection time, can quickly detect 5000-; standardized diagnostic standards exist to measure the degree of DNA fragmentation.

(2) Sperm Chromatin Dispersion (SCD): the method is a standard method for detecting DFI before the invention of flow cytometry, has complex operation process and long time, and depends on subjective judgment.

(3) Comet test: quantitative detection, counting of 200-500 sperms by manual observation each time, long detection time, complex method, strong subjectivity and difficult standardization.

(4) End Labeling (TUNEL): can detect apoptosis sperms, and is widely used for the research of apoptosis due to sensitivity and reliability. However, the TUNEL test is to establish a standardized method and is difficult to be used for routine detection of clinical sperms.

(5) Enzyme-linked immunoassay for 8-hydroxydeoxyguanosine (8-OHdG): has higher sensitivity, small sample amount, non-damage, rapidness and good selectivity. This method has some disadvantages, such as incomplete enzymatic digestion of DNA, which may result in a higher detection value than the true value.

(6) Fluorescence in situ labeling method: the method is direct, objective, efficient and rapid, but has narrow application, and is mainly used for chromosome euploid detection.

The advantages and disadvantages of the above technologies are compared as follows:

serial number	Product/technology name	Principle of principle	Is characterized in that
				1	Analysis of Sperm Chromatin Structure (SCSA)	Acridine orange emits different fluorescence after being combined with different DNA in a flow mode And counting by a cytometer.	Simple, efficient, rapid, objective, stable and easy to standardize, and is the best technique which can be provided for clinic at present And (4) performing the operation.
2	Sperm chromatin diffusion method (SCD)	Removing nucleoprotein after acid treatment of sperm DNA, staining with DAPI, etc The color was observed with a microscope.	Diffusion of nuclei was observed by naked eyeThe condition is slow, time-consuming and labor-consuming; the result of the detection being dependent on a person And (5) subjective judgment.
				3	COMET test (COMET)	Single cell gel electrophoresis	The sensitivity is high, but the establishment of a standardized method is difficult, and the application to clinic is difficult.
4	End labeling method (TUNEL)	With exogenous nucleotides in apoptotic cells catalyzed by enzymes Single-stranded or double-stranded.	The kit can detect apoptotic sperms, and is sensitive and reliable; however, it is difficult to establish a standardized method and to apply it to clinical applications.
				5	8-Hydroxydeoxyguanosine (8-OHdG) enzyme-linked Immunoassay method	Liquid chromatography, i.e. HPLC analysis of a sample hydrolysed by DNase 8-OHdG was quantitatively determined after isolation.	The sensitivity is higher; but incomplete enzymolysis of DNA exists, so that the detection value is higher than the true value; become into The method is high and the accuracy is low.
6	Fluorescence in situ hybridization technique (FISH)	And (4) probe hybridization.	Chromosome aneuploidy can be detected, and the detection of DNA fragments is difficult.

It can be seen that compared with other sperm DNA integrity detection methods, the flow cytometry-based SCSA method has the advantages of quantitative detection, high sensitivity, simple operation method, greatly shortened detection time due to the rapid detection capability of the flow cytometer, direct, objective and accurate data analysis, and has become the first choice for sperm DNA integrity detection at present. The method is based on acridine orange detection method, wherein acridine orange combines with single-chain or double-chain DNA to generate different fluorescence, and sperm DNA fragment rate value is calculated by identifying fluorescence color and counting. However, when sperm DNA is detected by the conventional flow cytometry, RNA in the sperm is also in a single-stranded state, and is also bound with acridine orange, and the emitted fluorescence is the same as the fluorescence emitted by the acridine orange/single-stranded DNA binding, so that the DNA detection signal contains an RNA detection signal, and the detection result cannot comprehensively, accurately and truly reflect the condition of the sperm DNA.

Therefore, there is a need to improve the detection method of sperm DNA fragmentation rate and develop related products.

Disclosure of Invention

In order to solve the above problems, in one aspect, the present application provides a method for detecting a DNA fragmentation rate of sperm based on flow cytometry, comprising the step of subjecting a sperm sample to a digestion treatment using ribonuclease.

In specific embodiments, the ribonuclease is dnase-free ribonuclease a (rnase a).

In a specific embodiment, the ribonuclease is present at a concentration of 0.01-1 mg/mL. In another specific embodiment, the ribonuclease is present at a concentration of 0.1 mg/mL.

In a particular embodiment, the temperature of the digestion treatment is between 15 and 70 ℃. In another specific embodiment, the temperature of the digestion treatment is 37 ℃.

In a particular embodiment, the digestion treatment time is 3 to 10 minutes. In another specific embodiment, the time of the digestion treatment is 5 minutes.

In a specific embodiment, the methods provided herein further comprise diluting the sperm sample to 1-2X 10 with a dilution buffer prior to the digestion process⁶And (4) step of grinding the powder into a powder/mL.

In specific embodiments, the dilution buffer is a mixture of: 0.01mol/L Tris-HCl (Tris-HCl), 0.15mol/L NaCl (NaCl), 1mmol/L EDTA (ethylenediaminetetraacetic acid) and 0.1% Triton-X100 (polyethylene glycol octylphenyl ether); the pH was 7.4.

In a specific embodiment, the methods provided herein further comprise the step of treating the sperm sample with an acidification buffer after the digestion treatment, and further adding acridine orange staining solution for staining.

In specific embodiments, the acidification buffer is a mixture of: 0.08 mol/L HCl (hydrochloric acid), 0.15mol/L NaCl (sodium chloride) and 0.1% (polyoxyethylene octylphenyl ether); the pH was 1.2.

In specific embodiments, the acridine orange staining solution is a mixture of: 0.1mol/L citric acid, 0.2mol/L Na₂HPO₄(disodium hydrogen phosphate), 1mmol/L EDTA, 0.15mol/L NaCl and 6. mu.g/mL acridine orange.

In particular embodiments, the methods provided herein further comprise the step of detecting the stained sperm sample using a flow cytometer.

In particular embodiments, the flow cytometer records at least a main population of 5000 cells of the sample at the time of detection.

In another aspect, the present application provides a kit for flow cytometry-based detection of sperm DNA fragmentation rate, comprising: a dilution buffer which is a mixture of: 0.01mol/L Tris-HCl, 0.15mol/L sodium chloride, 1mmol/L sodium ethylene diamine tetracetate and 0.1% Triton-X100, and the pH value is 7.4; ribonuclease A at a concentration of 10-100mg/mL, stored at-20 ℃ in a buffer of 50mmol/L Tris-HCl (pH 7.4) and 50% (v/v) glycerol

Performing the following steps; an acidification buffer, which is a mixture of: 0.08 mol/L hydrochloric acid, 0.15mol/L sodium chloride and 0.1% polyethylene glycol octyl phenyl ether, wherein the pH value is 1.2; and acridine orange staining solution which is a mixture of: 0.1mol/L citric acid, 0.2mol/L disodium hydrogen phosphate, 1mmol/L ethylene diamine tetraacetic acid, 0.15mol/L sodium chloride and 6 mu g/mL acridine orange. In specific embodiments, the ribonuclease a does not contain dnase.

In a specific embodiment, the ribonuclease A is used at a final concentration of 0.01-1mg/mL, preferably 0.1 mg/mL.

In a specific embodiment, the RNase A is used at a temperature of 15-70 deg.C, preferably 37 deg.C.

In a specific embodiment, the ribonuclease A is used for a period of 3 to 10 minutes, preferably 5 minutes.

In still another aspect, the application of the kit of the present application in detecting sperm DNA fragmentation rate is provided.

Therefore, by detecting the DNA fragmentation rate (DFI) of the sperms after eliminating the RNA in the sperms, the detection result is more accurate, the condition of the DNA of the sperms can be reflected more truly, and the characteristics of rapidness, simplicity, convenience, high efficiency, high flux, strong repeatability and the like of the detection method are not influenced.

Drawings

FIG. 1 is a diagram of a sperm cell population, wherein the central region of the gate circle is used to identify the primary population of sperm cells.

FIG. 2 is a fluorescence expression diagram of a sperm cell population divided into three groups of cells, wherein the cell population near FITC (vertical axis) is a cell population with good DNA integrity, the cell population near PerCP (horizontal axis) is a cell population with poor DNA integrity, and the cell population between the two is a cell population with poor DNA integrity.

Detailed description of the preferred embodiments

In order to more accurately determine the sperm DNA fragmentation rate, the inventor of the application finds out through deep research that the negative influence of a fluorescence signal emitted after RNA in sperm is combined with acridine orange is eliminated, and the accuracy of a detection result can be obviously improved, so that the sperm DNA fragmentation rate detection method after RNA elimination is established.

Specifically, the method for detecting the sperm DNA debris after RNA elimination provided by the application comprises the following steps:

(1) diluting the liquefied semen to 1-2 multiplied by 10 by using a 37 ℃ dilution buffer solution⁶Per mL;

(2) adding 500 mu L of diluted sperm into a flow cytometer loading tube;

(3) adding 5 mu L of RNase, and digesting for 5 minutes;

(4) adding an acidification buffer solution (the main component of the acidification buffer solution is hydrochloric acid, aiming at loosening the compact DNA structure so as to facilitate the subsequent dyeing step), adding acridine orange dyeing solution after 30 seconds, dyeing for 1 minute, and performing detection on a machine;

(5) setting the calibration of a flow cytometer, installing a detection tube on the machine, continuously measuring each tube of sample at least twice, recording at least 5000 cells of a sample main group in each tube of sample, and performing statistical analysis by using sperm DNA fragment rate flow software;

(6) collecting red and green fluorescence signals (namely FITC and PerCP fluorescence signals) of the sperms from the dyed sperm cell suspension by a flow cytometer, and judging the DNA fragmentation degree of the sample according to the proportion of the collected red fluorescence sperms to the total number of the collected sperms; the result judgment is based on the established image collection template, mainly based on the requirement on the fluorescence intensity (establishing corresponding templates according to different collection conditions of different instruments, specifically including, taking Calibur instrument as an example, the FITC fluorescence intensity median in the middle of the collection range is 400-600, and the PerCP fluorescence intensity median is 50-100;

(7) after the measurement, cell debris and fluorescent dyes remaining in the flow cytometer sample line were thoroughly removed with bleach, a sample tube cleaner, and sterilized double distilled water, respectively.

The dilution buffer solution in the step (1) is a mixture of 0.01mol/L Tris-HCl, 0.15mol/L NaCl (sodium chloride), 1mmol/L EDTA (ethylene diamine tetraacetic acid) and 0.1% Triton-X100, and the pH value is 7.4.

The RNase in the step (3) is RNase A without DNase, the concentration is 10-100mg/mL, the RNase A is stored in 50mmol/L Tris-HCl (pH 7.4) and 50% (v/v) glycerol at the temperature of-20 ℃, and the RNase A is used for digesting RNA in sperms and avoiding red fluorescence after single-stranded RNA staining to influence the accuracy of a DNA detection result.

The acidification buffer solution in the step (4) is 0.08 mol/L hydrochloric acid, 0.15mol/L NaCl and 0.1% Triton-X100.

Step (ii) of(4) The acridine orange staining solution is 0.1mol/L citric acid and 0.2mol/L Na₂HPO₄(disodium hydrogen phosphate), 1mmol/L EDTA, 0.15mol/L NaCl and 6. mu.g/mL acridine orange. After acidification treatment, the fragmentation degree of sperm DNA is determined by utilizing the characteristic that acridine orange dye is combined with double-stranded DNA to emit green or yellow fluorescence and is combined with single-stranded DNA to emit red fluorescence.

Therefore, the sperm DNA fragmentation rate detection method provided by the application treats and dyes the sperm sample, digests RNA in the sperm, and detects the sperm DNA fragmentation rate through fluorescence labeling and a flow type computer, so that the DNA damage degree of the sperm is truly evaluated, and the method provides help for infertility examination and selection of an auxiliary reproductive treatment scheme. The method avoids signals caused by fluorescence emitted by combining RNA in the sperms with acridine orange, has more accurate detection results, and realizes the purposes of simple, quick, accurate and high-throughput detection of the sperm DNA fragmentation rate.

On the basis, the application also provides a corresponding kit for detecting the DNA fragmentation rate of the sperms and application thereof.

For a better understanding of the present disclosure, reference will now be made to the following detailed description taken in conjunction with the accompanying drawings. It should be understood that these examples are for further illustration only and are not intended to limit the scope of the present application. In addition, after reading the contents of the present application, some insubstantial changes or modifications of the present application will still fall within the scope of the present application.

The reagents, materials and methods in the following examples are, unless otherwise indicated, all conventional in the art.

Example 1

This example provides the use of the method of the invention to detect sperm DNA fragmentation rate, wherein the RNase enzyme digestion assay comprises the following steps:

1.1 preparation of the article:

sterile disposable plastic products (centrifuge tubes and tip heads), Trizol reagent, chloroform, isopropanol, 75% ethanol, RNase-free water, RNase A and a nucleic acid quantitative detector are prepared.

1.2 extraction and separation of sperm cell total RNA:

1.2.1 lysis of sperm cells (cell size 5X 10)⁸One)

The liquefied semen was centrifuged at 800g for 10 minutes at room temperature, and the supernatant was discarded completely. Add 1mLTrizol and mix by pipetting 5 times immediately.

1.2.2 standing at room temperature for 5 minutes. An additional 0.25mL of chloroform was added and the tube was inverted vigorously to mix. After standing for 5 minutes, the mixture was centrifuged at a centrifugation speed of 12000g for 10 minutes at room temperature.

1.2.3 carefully transferring the upper aqueous phase to another centrifuge tube, adding 0.7 times volume of isopropanol, and shaking to mix. The mixture was left at room temperature for 10 minutes.

1.2.4 centrifugation at 12000g for 10min at room temperature. The supernatant was carefully discarded.

1.2.5 Add 500. mu.L of 75% ethanol to the centrifuge tube, shake for a moment, and centrifuge for 5 minutes. The supernatant was carefully discarded.

1.2.6 standing at room temperature for 15 minutes the RNA precipitate was just dried.

1.2.7 Add 120. mu.L of water to dissolve the RNA.

1.2.8 mixing, collecting 2 μ L, and detecting A with nucleic acid quantitative detector₂₆₀Values RNA content was calculated (see table below).

1.2.9 the remaining RNA was divided into 3 portions (22, 55. mu.L, respectively): the method is respectively used for detecting the digestion effect of RNA under different temperatures, different digestion times and different RNase concentrations.

1.2.9.1 mu.L of 10mg/mL RNase was added to the first RNA, mixed, digested at room temperature for 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 minutes, and 2. mu.L of the RNA was removed and assayed using a nucleic acid quantitative analyzer (see Table below).

1.2.9.2 mu.L of 10mg/mL RNase was added to the second RNA, mixed, digested at 37 ℃ for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 minutes, and 2. mu.L of the resulting mixture was removed and the RNA content was determined using a nucleic acid quantitative analyzer (see Table below).

1.2.9.3 the third RNA was further divided into 10 portions (5.4. mu.L each), 0.6. mu.L of RNase (0.01 mg/mL, 0.05mg/mL, 0.1mg/mL, 0.25mg/mL, 0.5mg/mL, 0.75mg/mL, 1.0mg/mL, 2.5mg/mL, 5mg/mL, 7.5mg/mL, 10 mg/mL) was added thereto, and after mixing, the mixture was digested at 37 ℃ for 5 minutes, 2. mu.L of RNase was removed therefrom, and the RNA content was measured with a nucleic acid quantitative analyzer (see Table below).

1.2.10 according to the results of the following Table for RNA content, when the RNA content is essentially undetectable (< 0.001. mu.g/mL), the preferred final RNase concentration is 0.1mg/mL, the preferred digestion time is 5 minutes, and the effect at 37 ℃ digestion temperature is better than that at room temperature.

1.2.11 the RNA content of the nucleic acid quantitative detector is shown in the following table:

example 2

This example provides for the detection of sperm DNA fragmentation rates using various methods.

Specifically, the method comprises the following steps:

2. preparation of samples

2.1 dividing 20 parts of naturally liquefied semen (each sample volume is 1.5-5.5mL, sperm concentration is 1500-: the method A comprises the following steps: flow cytometry of digested RNA for DFI; the method B comprises the following steps: flow cytometry detection of DFI after undigested RNA; the method C comprises the following steps: detecting DFI by a sperm chromatin diffusion method;

2.2 method A detection of DFI:

2.2.1 diluting the sperm with a buffer at 37 ℃ to 1-2X 10⁶Per mL; wherein the buffer solution is a mixture of 0.15mol/L NaCl (sodium chloride) and 1mmol/L EDTA (ethylene diamine tetraacetic acid), and the pH value is 7.4;

2.2.2 mu.L of diluted sperm was added to the flow cytometer loading tube, 5. mu.L of 10mg/mL RNase A (purchased from Sigma) was added, and digested at 37 ℃ for 5 minutes;

2.2.3 later, 400. mu.L of acidified buffer 0.08 mol/L hydrochloric acid was added, the mixture was placed on ice in an ice box, and after 30 seconds, staining solution (0.1mol/L citric acid, 0.2mol/L Na) was added₂HPO₄(disodium hydrogen phosphate),a mixture of 1mmol/L EDTA, 0.15mol/L NaCl, 6. mu.g/mL acridine orange);

2.2.4 set the parameters of the BD FACSCalibur flow cytometer, turn on the 488 nm argon ion laser, adjust the voltage of the following 4 channels and collect the signals: SSC, FSC, FL1 (wavelength 515 and 545 nm), FL3 (the peak of the fluorescence spectrum falls in the deep red range, the wavelength is 650 nm);

2.2.5 putting the sample loading tube on the machine, making the stained sperm cell suspension pass through a flow cytometer, and setting the flow rate of the sample as MED (35 muL/min); SSC, FSC and red-green fluorescence signals (namely FITC and PerCP fluorescence signals) of the sperms are collected, and the collection mode is that X-axis and Y-axis parameters are preset to be FSC-H1024, SSC-H1024, FL 1-H1024 and FL 3-H1024;

2.2.6 each tube of samples is continuously measured at least twice, each tube of samples at least records 5000 cells of the main group of the samples, and the results are statistically analyzed by using FACSCalibur flow software; the DNA fragmentation degree of the sample is determined by the ratio of the red fluorescent sperm collected by the flow cytometer to the total number of collected sperm. The result judgment is based on the established image collection template, mainly based on the requirement on the fluorescence intensity, and corresponding templates are established according to different collection conditions of different instruments, and the FITC fluorescence intensity median of the BD FACSCalibur instrument is in the middle of the collection range and is 400-600; the median of the PerCP fluorescence intensity is 50-100;

2.2.7 sperm cell populations the area in the door circle is shown in FIG. 1, where the main sperm cell population is the area in the door circle. The fluorescence expression pattern of the sperm cell population is shown in fig. 2, wherein the sperm cell population is divided into three groups of cells, the cell population near FITC (vertical axis) is a cell population with good DNA integrity, the cell population near PerCP (horizontal axis) is a cell population with poor DNA integrity, and the cell population between the two is a cell population with poor integrity;

2.2.8 after the measurement, cell debris and fluorescent dyes remaining in the flow cytometer sample line were thoroughly removed with bleach, tube cleaner and sterilized double distilled water, respectively.

2.3 method B detection of DFI:

2.3.1 dilution of sperm with 37 ℃ buffer to 1-2X 10⁶Per mL; wherein the buffer solution is 0.15mol/L NaCl (sodium chloride), 1mmol/L EDTA (ethylene diamine tetraacetic acid) mixture, pH 7.4;

2.3.2 the following detection procedure is as in 1.2.3-1.2.8.

2.4 method C detection of DFI:

(in the method, sperms are mixed in agarose and are subjected to denaturation and cracking to remove nucleoprotein, sperm chromatin passes through a nuclear membrane and a plasma membrane and surrounds the sperms, a cotton wool or halo-like structure is formed around the sperms, and then dyeing is carried out by Swiss-Giemsa dye liquor, the sperms with complete DNA can generate large halos or middle halos, and the sperms with DNA fragments are small halos or no halos. the judgment standard is that the ratio of the corona to the sperm head is more than 2/3 and is a large corona, the ratio of the corona to the sperm head is more than 1/4 and is less than or equal to 2/3 and is a medium corona, and the ratio of the corona to the sperm head is less than or equal to 1/4 and is a small corona.

2.4.1 the relevant solutions were prepared and prepared according to the following table: storing at 2-8 deg.C in dark.

Composition of	Loading capacity	Composition (I)
			Solution 1	20 mL	0.01mol/L Tris-HCl (Tris-HCl), 0.15mol/L NaCl (NaCl), 1mmol/L EDTA (ethylenediaminetetraacetic acid)
Solution 2	700μ L	1.0% Low melting agarose
			Solution 3	20mL	0.08 mol/L hydrochloric acid
Solution 4	20mL	0.2mol/L DTT (dithiothreitol), 1mmol/L EDTA.2Na (disodium ethylenediamine tetraacetate), 2.5mol/L NaCl (sodium chloride), 0.2mol/L Tris (Tris (hydroxymethyl aminomethane), 1% Triton X-100、0.05% Tween-20
			Solution 5	20mL	2.0g/L of Rayleigh dye, 2.0g/L of giemsa dye, 0.1 g/L of methylene blue and 20 ml/L of glycerin in methanol
Solution 6	20mL	0.3g/L KH₂P0₄(Potassium dihydrogen phosphate) and 0.2g/L Na₂HPO₄(disodium hydrogen phosphate)
			Glass carrier Sheet	10 pieces	Coated 0.65% agarose

2.4.2 preparation of instruments and consumables:

apparatus for use in an operating process, comprising: a common optical microscope, a 80 ℃ constant-temperature water bath box, a 2-8 ℃ and-20 ℃ refrigerator, a constant-temperature heater, a palm centrifuge and the like; other desirable materials include: 22mm cover glass, a semen sampling device, disposable gloves, 1-5 mul adjustable pipette and disposable suction head, 20-200 mul adjustable pipette and disposable suction head, 1.5mL EP tube, 3-5 mL suction tube, forceps, dyeing plate, purified water, 70%/90%/100% ethanol solution, etc.

2.4.3 adjusting the room temperature to about 20-24 ℃ before detection;

2.4.4 adjusting the concentration of the sperms to 5-10 multiplied by 106/mL by using the solution 1;

2.4.5 placing the centrifuge tube containing the solution 2 at 80 ℃ for incubation for 5 minutes, after completely melting, centrifuging by a palm centrifuge, and placing on a metal constant temperature heater at 37 ℃ for standby (after transferring from 80 ℃ to 37 ℃ and balancing for at least 5 minutes, the centrifuge tube can be used);

2.4.6 taking sperm with the concentration of 5-10 multiplied by 10⁶Adding 30 mu L of the treated sample into the solution 2 (keeping constant temperature at 37 ℃ on a metal constant temperature heater), fully mixing uniformly, and incubating at 37 ℃ for later use;

2.4.7 mu.L of the sperm suspension prepared in the previous step was added immediately to the slide coated area. Quickly covering a cover glass sheet (without applying pressure to the cover glass sheet) which is precooled for 5min at the temperature of 2-8 ℃ in a refrigerator, and avoiding generating bubbles as much as possible; standing at 37 ℃ for 2min, and standing in a refrigerator at 2-8 ℃ for 5min to solidify;

2.4.8 the slide was removed from the refrigerator and carefully translated to remove the coverslip (during the removal of the coverslip, the coverslip was always sliding against the plane of the slide, and could not be lifted up off the plane of the gel).

2.4.9 solution 4 was preheated to 25 ℃ in advance; sucking 1mL of solution 3, dripping the solution on a glass slide, and incubating for 7 min; drained and 1 washing with ddH2O

2.4.10 suck 1mL of solution 4 and drip on the glass slide, incubate for 25 min; draining, washing with ddH2O for 10min × two times

2.4.11 draining, and dripping 70% ethanol for 2 min; draining, and dripping 90% ethanol for 2 min; draining, and dripping 100% alcohol; 2 min; draining, and naturally drying in the air;

2.4.12 dropping solution 5 (about 0.5-0.8 mL) on the glass slide, and staining for 1 min;

2.4.13 and then the solution 6 is dripped on the upper surface (the dripping amount is 2-3 times of that of the solution 1), and the two solutions are fully mixed by blowing out breeze with an aurilave, and dyeing is carried out for 3-10 minutes.

2.4.14 quick rinsing with tap water (the dye solution cannot be poured out first during rinsing, and should be rinsed with running water to prevent sediment from depositing on the specimen), and drying.

2.4.15 microscopic examination, taking a plurality of visual fields, counting more than 200 sperms, and calculating the DNA fragmentation rate of the sperms.

2.5 DFI detection by three methods is shown in the following table:

2.6 by adopting pairwise mean pairing t test, finding:

method a was statistically different compared to method B (p =0.005<0.05);

method a was not statistically different compared to method C (p =0.546>0.05);

method B was statistically different (p <0.05) compared to method C;

statistical results show that the DFI detected by the method A after the RNA enzyme digestion is consistent with the result of the method C; also, the DFI results of method A were smaller than those of method B, and the presence of RNA therein was presumed to be responsible for the higher DFI results.

The above description is not intended to limit the present application, nor is the present application limited to the above examples. Those skilled in the art should also realize that such changes, modifications, additions and substitutions are within the spirit and scope of the present application.

Claims

1. A method for detecting the DNA fragmentation rate of sperm comprising the step of subjecting a sperm sample to a digestion process using a ribonuclease.

2. The method according to claim 1, wherein the ribonuclease is DNase-free ribonuclease A, preferably at a concentration of 0.01-1mg/mL, more preferably 0.1 mg/mL.

3. A method according to claim 1 or 2, wherein the temperature of the digestion treatment is 15-70 ℃, preferably 37 ℃; the time is 3 to 10 minutes, preferably 5 minutes.

4. The method of any one of claims 1 to 3, wherein the method further comprises using dilution prior to digestion treatmentDiluting the sperm sample to 1-2 x 10 by using a buffer solution⁶A step of drying the cells/mL; wherein the dilution buffer is a mixture of: 0.01mol/L Tris-HCl, 0.15mol/L sodium chloride and 1mmol/L EDTA, and the pH value of the dilution buffer is 7.4.

5. The method according to any one of claims 1 to 4, wherein the method further comprises a step of treating the sperm sample with an acidification buffer solution after the digestion treatment, and further adding an acridine orange staining solution for staining; wherein the acidification buffer is a mixture of: 0.08 mol/L hydrochloric acid, 0.15mol/L sodium chloride and 0.1% Triton-X100, and the pH value of the acidification buffer is 1.2; the acridine orange staining solution is a mixture of: 0.1mol/L citric acid, 0.2mol/L disodium hydrogenphosphate, 1mmol/L EDTA, 0.15mol/L sodium chloride and 6. mu.g/mL acridine orange.

6. The method of any one of claims 1 to 5, wherein the method further comprises the step of detecting the stained sperm sample using a flow cytometer; preferably, at least 5000 cells of the main population of the sample are recorded at the time of detection.

7. A kit for detecting sperm DNA fragmentation rate comprising the following components:

-a dilution buffer, which is a mixture of: 0.01mol/L Tris-HCl, 0.15mol/L sodium chloride and 1mmol/L EDTA, and the pH value is 7.4;

ribonuclease at a concentration of 10-100mg/mL, stored in buffer at-20 ℃; the buffer is 50mmol/L Tris-HCl (pH 7.4) and 50% (v/v) glycerol;

-an acidification buffer, which is a mixture of: 0.08 mol/L hydrochloric acid, 0.15mol/L sodium chloride and 0.1% Triton-X100, and the pH value is 1.2; and

-acridine orange staining solution, which is a mixture of: 0.1mol/L citric acid, 0.2mol/L disodium hydrogenphosphate, 1mmol/L EDTA, 0.15mol/L sodium chloride and 6. mu.g/mL acridine orange.

8. The kit of claim 7, wherein the ribonuclease is DNase-free ribonuclease A.

9. The kit according to claim 7 or 8, wherein the ribonuclease a is used at a final concentration of 0.01-1mg/mL, preferably 0.1 mg/mL; the use temperature is 15-70 ℃, preferably 37 ℃; the application time is 3-10 minutes, preferably 5 minutes.

10. Use of a kit according to any one of claims 7 to 9 for detecting sperm DNA fragmentation rate.