CN114088491A - Sperm DNA fragment detection kit and detection method thereof - Google Patents
Sperm DNA fragment detection kit and detection method thereof Download PDFInfo
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- G—PHYSICS
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- 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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
A sperm DNA fragment detection kit, comprising: pre-coating a glass slide, low-melting-point gel, a denaturing solution, a lysis solution, a Wright stain and a buffer solution; the method is characterized in that the cracking solution contains a reducing agent tris (2-carboxyethyl) phosphine hydrochloride. The lysis solution contains tris (2-carboxyethyl) phosphine hydrochloride as a reducing agent, can efficiently reduce and open disulfide bonds contained in protamine for packaging sperm DNA, does not generate pungent smell, reduces potential health hazards to users, avoids the use of devices such as exhaust hoods and the like, and improves the working environment of the users; the use concentration of the tris (2-carboxyethyl) phosphine hydrochloride is remarkably low, so that remarkable economic benefits of greatly reducing the cost of materials for production and use are brought.
Description
Technical Field
The invention belongs to in-vitro detection reagents, and particularly relates to a sperm DNA fragment detection kit and a detection method thereof.
Background
The degree of sperm DNA fragmentation is an important indicator of the integrity of the genetic material carried by male sperm. Abnormal degrees of male sperm DNA fragmentation are a common cause of male infertility. The detection methods widely applied at present mainly comprise a sperm chromatin structure analysis method (SCSA), a sperm chromatin diffusion method (SCD) and the like. Sperm chromatin spreading is an in situ detection method performed on slides. The detection principle is that sperms with more DNA double-strand breaks due to the influence of congenital or acquired factors are easily melted at double-strand break sites to generate single-strand DNA after being treated by a DNA denaturation solution, and then are mutually combined with a nuclear matrix. Under high salt and reducing conditions, DNA is detached from protamine, but cannot diffuse to form chromatin halos due to the presence of single-stranded DNA. While sperm with intact DNA can produce chromatin halos. The existence and size of the halo can be shown by a proper chromatin staining method, and the proportion of the number of the sperm without the halo or with smaller halo to the total number of the sperm can be calculated according to a determined standard so as to calculate the DNA fragment proportion (DFI) of the sample. The sperm chromatin diffusion method is a simple and rapid method for detecting the level of sperm DNA fragments.
Jose Luis Fern-ndez et al J Androl 2003; 24:59-66 for the first time, methods for detecting the level of sperm DNA debris using a sperm chromatin dispersion method are disclosed. The denaturation solution used in the method is 0.08mol/L (M) HCl, and the subsequent lysis steps are sequentially completed in two solutions, wherein one solution comprises 0.4M Tris (hydroxymethyl) aminomethane (Tris), 0.8M Dithiothreitol (DTT), 1% Sodium Dodecyl Sulfate (SDS), 50mM Ethylene Diamine Tetraacetic Acid (EDTA) and pH is 7.5; the second contained 0.4M Tris, 2M NaCl, 1% SDS, pH 7.5. Based on the improved sperm chromatin spreading method described above, Halotech markets a sperm DNA fragmentation detection kit under the trade names Halosperm and Halosperm G2.
The method has the following defects:
first, the presence of dithiothreitol in high concentrations in the lysate, which can lead to various disadvantages. The concrete expression is as follows: 1) since the implementation process comprises a large number of links of lysate exposed to air, dithiothreitol can generate strong pungent smell. Such an odor of dithiothreitol may be harmful to health if inhaled by a human body, and precautions such as using a fume hood, an exhaust hood, etc. must be taken to avoid inhalation. 2) Dithiothreitol is easily oxidized by oxygen in the air, and in practical application, along with the repeated use and continuous contact of a user with the air, the reducibility is gradually lost, and the cracking effect of the lysate is weakened. 3) Dithiothreitol can exert its reducibility only in the solution environment with pH > 6.5. The acidic hydrochloric acid solution providing the DNA denaturation condition is often partially brought into the subsequent lysis solution along with the sample slide, so that the pH of the lysis solution is reduced, and the reducibility of dithiothreitol in the lysis solution is influenced. 4) As a relatively expensive compound, high concentrations of dithiothreitol also result in relatively high cost of the reagents used in the assay.
Chapel Jing et al in the journal of China Male science 2009,15 (12): 1116-1119 discloses an improved test method for sperm chromatin diffusion. The lysate disclosed therein contains 0.4M Tris, 0.1M dithiothreitol, 0.8% sodium dodecyl sulfate, 40mM EDTA. The method reduces the use concentration of dithiothreitol, and improves the experimental environment of technicians to a certain extent. However, the relatively low concentration of dithiothreitol in the method also affects the cracking efficiency correspondingly, so that the generated halo is not obvious, and the interpretation accuracy of the result is affected. Sodium Dodecyl Sulfate (SDS) easily damages the sperm tail and also affects the identification of cell types.
Second, the technical problem posed by the relatively fixed process framework. In summary, the detection process framework of the SCD method is based on a processing mode in which a sample to be examined is fixed on a slide and then sequentially immersed in different reagents. Some of the facts that accompany this mode of treatment, including the need for the user to use a container of some sort to hold a volume of different reagents, the conventional choice may be a different size coplin jar (coplin jar). For example, a small copulin staining jar, which can hold 5 slides, needs at least 20-25mL of reagent to fully immerse the area of the sample mounted on the slide. This means that even when one sample slide is examined, the same amount of reagent as that used for examining 5 sample slides needs to be used. These reagents, which come into contact and act on the sample, must be replaced and discarded after a period of time dictated by standard laboratory procedures. It is anticipated that this situation may occur in some small clinical testing laboratories. This results in a waste of reagents and increases the load on facilities for the harmless disposal of the waste reagents. Another fact is that when transferring reagents from original reagent bottles to copulin staining jars, and when transferring sample slides sequentially between different copulin staining jars, the reagents will inevitably come into contact with air, which means that the reagents will come into contact with oxygen in the air, and certain components of the reagents will evaporate into the air or release a certain odor. These effects are not negligible to the detector considering the large volume of reagent used per SCD method.
The sperm DNA fragmentation rate detection is a detection project widely developed in the reproductive health field, and imported and domestic sperm DNA fragmentation detection kits provided by different manufacturers are available in the market at present. The technical problems to be solved are ubiquitous, and the reagents have obvious pungent smell, are unstable, are high in cost and the like.
Disclosure of Invention
The invention aims to provide a sperm DNA fragment detection kit and a detection method thereof. The invention is based on the basic principle of a sperm chromatin diffusion method (SCD), overcomes peculiar smell interference and high cost caused by expensive Dithiothreitol (DTT) in a reagent in the prior art, and provides a sperm DNA fragment detection kit which has high efficiency, stability, environmental friendliness to a laboratory, low cost and flexible operation.
The purpose of the invention is realized as follows: a sperm DNA fragment detection kit comprising: pre-coating glass slides, low-melting-point gel, denaturing solution, lysis solution, Switzerland dye solution and buffer solution; the method is characterized in that the cracking solution contains a reducing agent tris (2-carboxyethyl) phosphine hydrochloride.
The object of the invention is also achieved in that: the concentration of the reducer tris (2-carboxyethyl) phosphine hydrochloride in the lysis solution is 1mM-500 mM.
The pre-coated glass slide is a glass slide coated with 0.1% -5% of agarose aqueous solution with the melting point of 20-60 ℃; the low-melting point gel is 0.1-5% of agarose hydrogel with a melting point of 20-65 ℃; the denaturant is an aqueous solution containing 0.08-0.1M HCl; the lysis solution is an aqueous solution containing 2-2.5M NaCl, 0.01-0.1M Tris (2-carboxyethyl) phosphine hydrochloride, 0.1-0.3M Tris, 0.5-2% Triton X-100, 0-20mM EDTA and pH 7.0-8.0; the Reye dye solution is a glycerol-containing methanol solution containing 0.05-0.5% of Reye pigment and 0.01-0.1% of Gerstmann pigment; the buffer solution is 0.01-0.1M phosphate buffer solution with the pH value of 6.0-7.0.
The pre-coated slide is prepared by the following method:
1) weighing 1-50g of low-melting-point agarose, placing in a 1L heat-resistant container, adding 50-200ml of pure water, and stirring in a water bath at 99 +/-1 ℃ to completely dissolve the agarose;
2) adding pure water to a constant volume of 1L, stirring and mixing uniformly, and keeping the temperature at 70 +/-5 ℃;
3) subpackaging a proper amount of the solution into a coplin jar, vertically immersing a clean glass slide into the completely melted agarose solution, standing for 1-30 seconds, lifting and immersing again to form a uniform film on the glass slide;
4) taking out the glass slide, wiping off agarose on the back surface by using absorbent paper, horizontally placing the glass slide on a smooth metal surface of a low-temperature (4 ℃) metal bath, and placing and cooling the glass slide to completely solidify the agarose; or the glass slide is horizontally placed at room temperature and naturally solidified;
5) the slide was placed horizontally in a drying oven or dry environment at a temperature range between room temperature and 100 ℃ until the agarose was completely dried and a fine film adhered to the glass was formed.
The low melting point gel is prepared by the following method:
1) weighing 1-50g of low-melting-point agarose, placing in a 1L heat-resistant container, adding 50-200ml of pure water, and stirring in a water bath at 99 +/-1 ℃ to completely dissolve the agarose;
2) adding pure water to a constant volume of 1L, stirring and mixing uniformly, and keeping the temperature at 45 +/-5 ℃;
3) the solution was split into 0.2ml sample tubes, 0.07ml per tube, while hot, to allow for natural clotting.
The denaturant is prepared by the following method:
1) putting 200 plus 500mL of pure water into a 1L volumetric flask, adding 6.7-8.3mL of concentrated hydrochloric acid into the volumetric flask, and stirring to uniformly mix the pure water and the concentrated hydrochloric acid;
2) adding pure water to a constant volume of 1L, and stirring and mixing uniformly.
The lysate is prepared by the following method:
1) placing 800-900ml pure water into a 1L volumetric flask, weighing 117-146.3g of NaCl, 12.1-36.3g of Tris, 2.9-28.7g of Tris (2-carboxyethyl) phosphine hydrochloride and 0-5.8g of EDTA, adding into the volumetric flask, and stirring to completely dissolve the materials;
2) measuring Triton X-1005-20 mL, adding into the volumetric flask, and stirring to completely dissolve;
3) slowly adding concentrated hydrochloric acid, and adjusting pH to 7.0-8.0;
4) the volume is adjusted to 1L by pure water.
The Reynold dye solution is prepared by the following method:
1) weighing 0.25-2.5g of Swiss pigment and 0.05-0.5g of Gerstmann pigment, putting the weighed materials into a clean mortar, adding 5-20mL of methanol and 1-4mL of glycerol, grinding for a moment, and sucking out upper dye liquor; adding 5-20ml of methanol, continuously grinding, sucking the supernatant out, and repeating the steps for several times until the dye is completely dissolved;
2) and (4) adding methanol to a constant volume of 500ml, stirring and mixing uniformly, and transferring to a brown reagent bottle for storage.
The buffer is prepared by the following method:
1) putting 50-200ml of pure water into a 1L volumetric flask, weighing 0.57-5.7g of disodium hydrogen phosphate and 0.82-8.2g of monopotassium phosphate, adding into the volumetric flask, and stirring to completely dissolve;
2) detecting the pH value of the solution by using a pH meter, and adjusting the pH value to 6.0-7.0;
3) the volume is adjusted to 1L by pure water.
The detection method of the sperm DNA fragment detection kit comprises the following steps:
(one) reagent preparation
1) Placing a sample tube pre-filled with 0.07ml of low-melting-point gel at 80-95 ℃ for incubation for 5-20 minutes, after complete melting, placing the sample tube at 37 ℃ for standby, and balancing for at least 5 minutes;
2) adjusting the room temperature to 20-25 ℃ before detection;
3) recovering the denatured liquid and the lysate to room temperature;
(II) sample preparation
1) Adjusting liquefied fresh semen or frozen and thawed semen with liquid nitrogen or separated motile semen to 5-10 × 10 with physiological saline6/ml；
(III) detection step
1) Adding 30 mu L of the sample to be detected with the concentration into the sample tube containing the melted low-melting-point gel, fully and uniformly mixing to prepare sperm suspension, and incubating at 37 ℃ for later use;
2) placing the pre-coated glass slide in a refrigerator at 2-8 ℃ for precooling for 5 minutes, and then taking out, or horizontally placing the pre-coated glass slide on a metal bath precooled at 4 ℃, and quickly adding 5-30 mu L of the sperm suspension into a glass slide coating area;
3) cover the cover glass rapidly immediately, avoid producing the bubble; placing the mixture in a refrigerator at the temperature of 2-8 ℃ for 5-10 minutes to completely solidify the mixture; or standing in a pre-cooled metal bath at 4 deg.C for 5-10 min to solidify completely;
4) taking out the glass slide, and removing the cover glass along the horizontal direction;
5) the glass slide is immediately and accurately reacted with the denaturation liquid for 7 minutes;
6) taking out the glass slide, and sucking out liquid remained on the back of the glass slide by using filter paper; accurately reacting the glass slide with the lysis solution for 25 minutes;
7) taking out the glass slide, and sucking out liquid remained on the back of the glass slide by using filter paper; vertically immersing the glass slide in a large amount of pure water for 5 minutes;
8) taking out the glass slide, and sucking out liquid remained on the back of the glass slide by using filter paper; vertically immersing the glass slide into a reaction tank filled with 70% ethanol for 2 minutes;
9) taking out the glass slide, and sucking out liquid remained on the back surface of the glass slide by using filter paper; vertically immersing the glass slide into a reaction tank filled with 100% ethanol for 2 minutes;
10) naturally drying or air drying in the air;
11) covering each glass slide with about 150 mu L of Swiss stain, slowly adding about 300 mu L of buffer solution after 1-2min, lightly blowing and beating the mixed solution by an ear washing ball, standing at room temperature for 15min, and then lightly washing the stained slide with running water for about 3-6 s;
12) naturally drying or blow-drying;
13) 500 sperm were observed under a 40 × objective microscope, and the criteria for judgment were: fragmented sperm are obtained when the width of the halo is less than or equal to one third of the smallest diameter of the sperm head, and intact sperm are obtained when the width of the halo is greater than one third of the smallest diameter of the sperm head. And calculating the proportion of the number of the sperm fragmented from the DNA in the total number of the sperm to be detected, namely the sperm DNA fragmentation rate of the sample.
One of the preferable schemes of the detection method of the sperm DNA fragment detection kit is characterized in that: the reaction mode of the detection step 5) is that the glass slide is horizontally placed, 2mL of denaturant liquid is immediately dripped into a sample gel area of the glass slide, the glass slide is inclined after reaction, and the liquid flowing down is sucked by filter paper; and the detection step 6) adopts a reaction mode that the glass slide is horizontally placed, 2mL of lysate is dripped in a sample gel area, the glass slide is inclined after reaction, and the liquid flowing down is sucked by filter paper.
The second preferred embodiment of the detection method of the sperm DNA fragment detection kit is characterized in that: wherein the reaction mode of the detection step 5) is that the glass slide is immediately and vertically immersed into a reaction tank containing the denaturation liquid; and the detection step 6) adopts a reaction mode that the glass slide is vertically immersed into a reaction tank filled with a lysis solution.
The invention has the following remarkable technical effects:
1. the lysis solution contains tris (2-carboxyethyl) phosphine hydrochloride as a reducing agent, can efficiently reduce and open disulfide bonds contained in protamine for packaging sperm DNA, does not generate pungent smell, reduces potential health hazards to users, avoids the use of equipment such as an exhaust hood and the like, and improves the working environment of the users; when the lysate using dithiothreitol as a reducing agent is subjected to operations similar to those described above, protective measures such as an exhaust hood are required due to the generation of pungent odor.
2. According to the preparation method of the invention, 10-25mM tris (2-carboxyethyl) phosphine hydrochloride can achieve the reduction effect of 200-800mM dithiothreitol, and the use concentration is obviously reduced. At the current commercial prices of chemicals, the two are within about 50%. For example, the price of 5g of reagent grade tris (2-carboxyethyl) phosphine hydrochloride (cat 580567) sold by MERCK company is 3214 yuan, and the price of 5g of molecular biology grade dithiothreitol (cat 43815) is 3966 yuan. But the use concentration of the tris (2-carboxyethyl) phosphine hydrochloride is obviously lower, so that the obvious economic benefit of greatly reducing the cost of materials for production and use is brought.
3. As is well known, tris (2-carboxyethyl) phosphine hydrochloride does not react with oxygen, and the lysis solution formed by using tris (2-carboxyethyl) phosphine hydrochloride as a reducing agent component is stable in the air, has long validity period and is easier to store in a production link or after being unsealed and used. Other prior art reducing agents such as dithiothreitol are susceptible to oxidation by air and gradual failure.
4. As a known reducing agent, tris (2-carboxyethyl) phosphine hydrochloride is also publicly described as having properties such that the reducibility of tris (2-carboxyethyl) phosphine hydrochloride under acidic conditions (pH >1.5) is not affected, whereas dithiothreitol requires a pH of more than 6.5 for its reducibility to develop. In sperm chromatin diffusion experiments, acidic solutions providing DNA denaturation conditions are often partially carried into subsequent lysates along with the sample slides, lowering the pH of the lysates, thereby significantly compromising the reduction effect of dithiothreitol. The invention takes tris (2-carboxyethyl) phosphine hydrochloride as a reducing agent, the performance of the prepared lysate is not influenced by the residual acidic denaturant in the previous step, the pH adaptation range is wide, and the effect is stable.
5. According to one of the preferable schemes of the detection method of the kit, the amount of the single-use reagent can be obviously reduced, the waste of the reagent is avoided, the cost is further reduced, and the load on facilities for harmlessly treating the waste reagent is reduced. The technical problem caused by a relatively fixed flow framework of the SCD method is solved.
6. By using the kit and the detection method, the chromatin halo generated by sperm with complete DNA can achieve or exceed the effect which is also obvious and easy to identify in the prior art.
Drawings
FIG. 1 is a microscope image showing the result of the examination in example 4 of the present invention.
FIG. 2 is an image of representative results generated using the kit containing tris (2-carboxyethyl) phosphine hydrochloride at various concentrations in example 5 and a commercially available sperm DNA fragment detection kit.
Detailed Description
The present invention is described in further detail below by way of specific examples.
The Chinese and English references of the terms used in the present invention are as follows:
tris (hydroxymethyl) aminomethane: tris (English and abbreviation, same below)
Dithiothreitol: DTT (data transfer technology)
Sodium lauryl sulfate: SDS
Ethylene diamine tetraacetic acid: EDTA
Tris (2-carboxyethyl) phosphine hydrochloride: TCEP HCl
Agarose: agarose
Triton (trade name): triton X-100
A sperm chromatin diffusion method: SCD
DNA fragment ratio: DFI
mol/L is abbreviated to M
In clinical examination work for detecting the DNA fragmentation degree of male sperms by using a sperm chromatin diffusion method, the inventor is satisfied with the characteristics of simplicity, rapidness, intuition and independence of complex instruments of a sperm chromatin diffusion method (SCD) method on one hand, and feels puzzled on various problems caused by using dithiothreitol as a reducing agent and a relatively fixed process framework of the SCD method on the other hand. The widespread use of dithiothreitol in the aforementioned prior art highlights the technical drawbacks of the SCD method. Dithiothreitol is a common reducing agent with sulfhydryl smell, can be oxidized by oxygen in the air, has a working pH range of 6.5-9.0, and is widely applied in the fields of molecular biology and the like. For example, 1mM DTT may be added to the buffer to protect the activity of some enzymes. In such applications, dithiothreitol is used in small concentrations and doses, and participates in a small volume closed-tube reaction, thus not causing significant odor distress to the user. In contrast, the higher concentrations (0.1-0.8M) and doses (>20mL) used made the above-mentioned intrinsic properties of Dithiothreitol (DTT) a drawback that is difficult to ignore in the flow-frame of the SCD method.
In response to the above technical problem, the inventors sought to find a DTT alternative that fits the framework of the SCD process flow, which can be broadly referred to as a reducing agent without irritating odor. By searching the literature, the inventors found that there was a reducing agent tris (2-carboxyethyl) phosphine hydrochloride, CAS number 51805-45-9, which is not irritating in odor. Through further experiments, the inventor surprisingly finds that tris (2-carboxyethyl) phosphine hydrochloride not only can be used as a substitute of dithiothreitol to realize the technical effect of the SCD method, but also can be matched with a system framework of a sperm chromatin diffusion method to comprehensively overcome other technical problems. The tri (2-carboxyethyl) phosphine hydrochloride has a plurality of properties which can show value in a sperm chromatin diffusion method, which shows the compatibility of the two. For example, pungent odor and a narrow pH range are negligible or easily circumvented for some experiments. And because the sperm chromatin diffusion method uses a large amount of lysate and requires a large amount of reducing agent, pungent odor is difficult to ignore, the cost is high, and the requirement on the efficiency of the reducing agent is high. Since the denaturing solution is acidic and inevitably brought into the lysis solution, the applicable pH range of the reducing agent is also important. These properties of tris (2-carboxyethyl) phosphine hydrochloride are known from published information. These very significant technical effects are obtained according to the invention by selecting tris (2-carboxyethyl) phosphine hydrochloride as reducing agent. Tris (2-carboxyethyl) phosphine hydrochloride is used as a reducing agent for a lysate in a sperm chromatin diffusion method, and reports on the sperm chromatin diffusion method in 2003 are not found so far. The inventor simultaneously finds that after the tris (2-carboxyethyl) phosphine hydrochloride is used for replacing dithiothreitol, a detection mode that a small amount of cracking reagent is dripped on a glass slide can be adopted, the purpose of obviously reducing the single use amount of the reagent is achieved, and therefore the problems of reagent waste, environmental protection and the like caused by a relatively fixed flow framework of an SCD method can be solved.
The sperm DNA fragment detection kit of the invention has the preferable scheme that the kit comprises: pre-coating glass slides, low-melting-point gel, denaturing solution, lysis solution, Switzerland staining solution and buffer solution; the pre-coated glass slide is a glass slide coated with 1% agarose aqueous solution with the melting point of 20-60 ℃; the low-melting point gel is 1% of agarose hydrogel with the melting point of 20-65 ℃; the denaturant is an aqueous solution containing 0.08-0.1M HCl; the lysis solution is an aqueous solution containing 2-2.5M NaCl, 0.025-0.05M Tris (2-carboxyethyl) phosphine hydrochloride, 0.2M Tris, 1% Triton X-100, 0-10mM EDTA and pH 7.0-7.5; the Swiss stain solution is a glycerol-containing methanol solution containing 0.1 percent of Swiss pigment and 0.03 percent of Gerstmann pigment; the buffer solution is 0.06M phosphate buffer solution with the pH value of 6.4-6.8.
The preferable concentration scheme of tris (2-carboxyethyl) phosphine hydrochloride contained in the lysate of the sperm DNA fragment detection kit is that the lysate contains numerical values of 0.01M, 0.015M, 0.02M, 0.025M, 0.03M, 0.035M, 0.04M, 0.045M, 0.05M, 0.055M, 0.06M, 0.065M, 0.07M, 0.075M, 0.08M, 0.085M, 0.09M, 0.095M and 0.1M.
In the sperm DNA fragment detection kit, the preferable concentration scheme of tris (2-carboxyethyl) phosphine hydrochloride contained in the lysate can also be 0.001M, 0.002M, 0.003M, 0.004M, 0.005M, 0.006M, 0.007M, 0.008M, 0.09M and 0.01M.
In the sperm DNA fragment detection kit, the preferable concentration scheme of tris (2-carboxyethyl) phosphine hydrochloride contained in the lysate can also be the numerical values of 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M and 0.5M.
Example 1
This embodiment provides a sperm DNA fragment detection kit, including: pre-coating glass slides, low-melting-point gel, denaturing solution, lysis solution, Switzerland dye solution and buffer solution;
the pre-coated glass slide is a glass slide coated with 1% agarose aqueous solution with the melting point of 20-60 ℃; the low-melting point gel is 1% of agarose hydrogel with the melting point of 20-65 ℃; the denaturant is an aqueous solution containing 0.1M HCl; the lysis solution is an aqueous solution containing 2.5M NaCl, 10mM Tris (2-carboxyethyl) phosphine hydrochloride, 0.2M Tris, 1% Triton X-100 and 5mM EDTA, and having a pH value of 7.0-7.5; the Swiss stain is a glycerol-containing methanol solution containing 0.1 percent of Swiss pigment and 0.03 percent of Gerstmann pigment; the Rui's buffer is 0.06M phosphate buffer with pH of 6.4-6.8.
The preparation method of the pre-coated glass slide comprises the following steps:
1) weighing 10g of low-melting-point agarose, placing the agarose in a 1L heat-resistant container, adding 200ml of pure water, and stirring in a water bath at 99 +/-1 ℃ to completely dissolve the agarose;
2) adding pure water to a constant volume of 1L, stirring and mixing uniformly, and keeping the temperature at 70 +/-5 ℃;
3) and (3) subpackaging a proper amount of the solution into a coplin jar, vertically immersing a clean glass slide into the completely melted agarose, standing for 2 seconds, lifting and immersing again to form a uniform film on the glass slide.
4) And taking out the glass slide, wiping agarose on the back surface of the glass slide by using absorbent paper, and horizontally placing the glass slide at room temperature for natural solidification.
5) The slide was placed horizontally in a room temperature dry environment until the agarose was completely dried and a fine film adhered to the glass was formed. The coated slide can be stored in a sealed condition at room temperature for several months. Complete drying of the agarose is important for the coating effect.
The preparation method of the low-melting-point gel comprises the following steps:
1) weighing 10g of low-melting-point agarose, placing the agarose in a 1L heat-resistant container, adding 200ml of pure water, and stirring in a water bath at 99 +/-1 ℃ to completely dissolve the agarose;
2) adding pure water to a constant volume of 1L, stirring and mixing uniformly, and keeping the temperature at 45 +/-5 ℃;
3) the solution was portioned while hot into 0.2ml sample tubes, 0.07ml per tube. After solidification, the mixture is stored at 4 ℃.
The preparation method of the denaturant comprises the following steps:
1) putting 500mL of pure water into a 1L volumetric flask, adding 8.3mL of concentrated hydrochloric acid into the volumetric flask, and stirring to uniformly mix the pure water and the concentrated hydrochloric acid;
2) adding pure water to a constant volume of 1L, and stirring and mixing uniformly.
The preparation method of the lysate comprises the following steps:
1) 900ml of pure water was put in a 1L volumetric flask, 146.3g of NaCl, 24.2g of Tris, 2.87g of Tris (2-carboxyethyl) phosphine hydrochloride and 1.46g of EDTA were weighed and added to the volumetric flask, and stirred to be completely dissolved.
2) Triton X-10010 mL was weighed out and added to the above volumetric flask, and stirred to dissolve completely.
3) Slowly adding concentrated hydrochloric acid, and adjusting pH to 7.0-7.5.
4) The volume is adjusted to 1L by pure water.
The preparation method of the Swiss stain comprises the following steps:
1) weighing 0.5g of Switzerland pigment and 0.15g of Gerstmann pigment, placing in a clean mortar, adding 20mL of methanol and 4mL of glycerol, grinding for a moment, and sucking out the upper dye solution; adding 20ml of methanol, continuously grinding, sucking the supernatant out, and continuing for several times until the dye is completely dissolved;
2) the volume is adjusted to 500ml by methanol, the mixture is evenly stirred and transferred to a brown reagent bottle for storage.
The preparation method of the Ruhry buffer solution comprises the following steps:
1) putting 200ml of pure water into a 1L volumetric flask, weighing 3.4g of disodium hydrogen phosphate and 4.9g of monopotassium phosphate, adding into the volumetric flask, and stirring to completely dissolve the disodium hydrogen phosphate and the monopotassium phosphate;
2) detecting the pH value of the solution by using a pH meter, and adjusting the pH value to 6.0-7.0;
3) the volume is adjusted to 1L by pure water.
Example 2
This embodiment provides a sperm DNA fragment detection kit, including: pre-coating glass slides, low-melting-point gel, denaturing solution, lysis solution, Switzerland dye solution and buffer solution;
the pre-coated glass slide is a glass slide coated with 1% agarose aqueous solution with the melting point of 20-60 ℃; the low-melting point gel is 1% of agarose hydrogel with the melting point of 20-65 ℃; the denaturant is an aqueous solution containing 0.1M HCl; the lysis solution is an aqueous solution containing 2.5M NaCl, 50mM Tris (2-carboxyethyl) phosphine hydrochloride, 0.2M Tris, 1% Triton X-100 and 5mM EDTA, and the pH value is 7.0-7.5; the Swiss stain solution is a glycerol-containing methanol solution containing 0.1 percent of Swiss pigment and 0.03 percent of Gerstmann pigment; the Rui's buffer is 0.06M phosphate buffer with pH of 6.4-6.8.
The preparation method of the lysate comprises the following steps:
1) 900ml of pure water was put in a 1L volumetric flask, 146.3g of NaCl, 24.2g of Tris, 14.3g of Tris (2-carboxyethyl) phosphine hydrochloride and 1.46g of EDTA were weighed and added to the volumetric flask, and stirred to be completely dissolved.
2) Triton X-10010 mL was weighed out and added to the above volumetric flask, and stirred to dissolve completely.
3) Slowly adding concentrated hydrochloric acid, and adjusting pH to 7.0-7.5.
4) The volume is adjusted to 1L by pure water.
The preparation methods of the pre-coated glass slide, the low-melting-point gel, the denaturation solution, the Reye staining solution and the Reye buffer solution are the same as those of the embodiment 1.
Example 3
This embodiment provides a sperm DNA fragment detection kit, including: pre-coating glass slides, low-melting-point gel, denaturing solution, lysis solution, Switzerland dye solution and buffer solution;
the pre-coated glass slide is a glass slide coated with 1% agarose aqueous solution with the melting point of 20-60 ℃; the low-melting point gel is 1% of agarose hydrogel with the melting point of 20-65 ℃; the denaturant is an aqueous solution containing 0.1M HCl; the lysis solution is an aqueous solution containing 2.5M NaCl, 100mM Tris (2-carboxyethyl) phosphine hydrochloride, 0.2M Tris, 1% Triton X-100 and pH 7.0-7.5; the Swiss stain solution is a glycerol-containing methanol solution containing 0.1 percent of Swiss pigment and 0.03 percent of Gerstmann pigment; the Rui's buffer is 0.06M phosphate buffer with pH of 6.4-6.8.
Example 4
This example provides detailed steps of a detection method of a sperm DNA fragment detection kit, as follows:
(one) reagent preparation
1) Placing a sample tube pre-filled with 0.07ml of low-melting-point gel at 80 ℃ for incubation for 20 minutes, after the low-melting-point gel is completely melted, placing the sample tube at 37 ℃ for standby application, and balancing for at least 5 minutes;
2) adjusting the room temperature to 22 ℃ before detection;
3) the denatured and lysed solutions were returned to room temperature.
(II) sample preparation
1) Adjusting liquefied fresh semen or frozen and thawed semen with liquid nitrogen or separated motile semen to 5-10 × 10 with physiological saline6/ml；
(III) detection step
1) Adding 30 mu L of the sample to be detected with the concentration into the sample tube containing the melted low-melting-point gel, fully and uniformly mixing to prepare sperm suspension, and incubating at 37 ℃ for later use;
2) placing the pre-coated glass slide in a refrigerator at 2-8 ℃ for precooling for 5 minutes, and then taking out, or horizontally placing the pre-coated glass slide on a metal bath precooled at 4 ℃, and quickly adding 5-30 mu L of the sperm suspension into a glass slide coated area;
3) cover the cover glass immediately, avoid producing the bubble; placing the mixture in a refrigerator at the temperature of 2-8 ℃ for 5-10 minutes to completely solidify the mixture; or left on a pre-cooled metal bath at 4 ℃ for 5-10 minutes to completely solidify.
4) Taking out the glass slide, and removing the cover glass along the horizontal direction;
5) horizontally placing the glass slide, immediately dropwise adding 2mL of denaturing solution into a sample gel area, and accurately reacting for 7 minutes; tilting the glass slide, and sucking the flowing liquid by using filter paper;
6) horizontally placing the glass slide, dropwise adding 2mL of lysate in a sample gel area, and accurately reacting for 25 minutes; tilting the glass slide, and sucking the flowing liquid by using filter paper;
7) vertically immersing the glass slide in a large amount of pure water for 5 minutes;
8) taking out the glass slide, and sucking out liquid remained on the back of the glass slide by using filter paper; vertically immersing the glass slide into a reaction tank filled with 70% ethanol for 2 minutes;
9) taking out the glass slide, and sucking out liquid remained on the back surface of the glass slide by using filter paper; vertically immersing the glass slide into a reaction tank filled with 100% ethanol for 2 minutes;
10) naturally drying or air drying in the air;
11) covering each glass slide with about 150 mu L of Reye staining solution, slowly adding about 300 mu L of Reye buffer solution after 1-2min, lightly blowing and beating the mixed staining solution by an aurilave, standing at room temperature for 15min, and lightly washing the staining slide with running water for about 3-6 s;
12) naturally drying or blow-drying;
13) 500 sperm cells were observed under a 20X or 40X objective microscope. Sperm heads with intact DNA produce large and medium halos, sperm heads with DNA debris present produce small halos or no halos. The criterion is whether the width of the halo exceeds one third of the smallest diameter of the sperm head. The proportion of the number of sperm with DNA debris in the total number of sperm examined is the sperm DNA debris rate of the sample.
The result image of the detection using the sperm DNA fragment detection kit of the present invention according to the protocol described in example 4 is shown in FIG. 1. The images show that sperm of DNA fragments are readily distinguishable from intact DNA sperm by halo size.
Example 5
This example compares the results of experiments using kits containing different concentrations of tris (2-carboxyethyl) phosphine hydrochloride and a commercially available sperm DNA fragment detection kit when following the experimental procedure described in example 4.
Kit 1: commercially available sperm DNA fragmentation detection kit (sperm chromatin dispersion method), medical device registration number: yue Ji Zhu Yu 20192400701. The main components are as follows: coating glass slide, fusible gel, reaction liquid A, reaction liquid B, Ruhrstan's stain, Ruhrstan's buffer solution, SCD preservation solution and cover glass.
And (3) kit 2: the kit described in example 1 contained tris (2-carboxyethyl) phosphine hydrochloride in the lysate in a concentration of 10 mM.
Kit 3: the kit described in example 2 contained tris (2-carboxyethyl) phosphine hydrochloride in the lysate in a concentration of 50 mM.
Kit 4: the kit described in example 3, the concentration of tris (2-carboxyethyl) phosphine hydrochloride contained in the lysate was 100 mM.
After the same sperm sample was tested using the four kits described above, the sperm DNA fragmentation rate (DFI) of the sample was counted. The sperm DNA fragmentation rate of the sample detected by the kit 1 is 18.3%, the sperm DNA fragmentation rate obtained by the kit 2 is 18.2%, the sperm DNA fragmentation rate obtained by the kit 3 is 18.14%, and the sperm DNA fragmentation rate obtained by the kit 4 is 18.02%.
The images of the test results generated using the four kits are shown in FIG. 2. The images show that the results of using the kit containing tris (2-carboxyethyl) phosphine hydrochloride with different concentrations all reach or exceed the effect of the commercial sperm DNA fragment detection kit.
Claims (13)
1. A sperm DNA fragment detection kit, comprising: pre-coating glass slides, low-melting-point gel, denaturing solution, lysis solution, Switzerland dye solution and buffer solution; the method is characterized in that the cracking solution contains a reducing agent tris (2-carboxyethyl) phosphine hydrochloride.
2. The sperm DNA fragment detection kit according to claim 1, wherein the lysis solution contains a reducing agent tris (2-carboxyethyl) phosphine hydrochloride at a concentration of 1mM-500 mM.
3. The sperm DNA fragment detection kit of claim 2, wherein the pre-coated slide is a slide coated with 0.1% -5% agarose aqueous solution having a melting point of 20-60 ℃; the low-melting point gel is 0.1-5% of agarose hydrogel with a melting point of 20-65 ℃; the denatured liquid is an aqueous solution containing 0.08M-0.1M HCl; the lysis solution is an aqueous solution containing 2-2.5M NaCl, 0.01-0.1M Tris (2-carboxyethyl) phosphine hydrochloride, 0.1-0.3M Tris, 0.5-2% Triton X-100, 0-20mM EDTA and pH 7.0-8.0; the Reye dye solution is a glycerol-containing methanol solution containing 0.05-0.5% of Reye pigment and 0.01-0.1% of Gerstmann pigment; the buffer solution is 0.01-0.1M phosphate buffer solution with the pH value of 6.0-7.0.
4. The sperm DNA fragment detection kit of claim 1, 2 or 3, wherein said pre-coated slide is prepared by the method of:
1) weighing 1-50g of low-melting-point agarose, placing in a 1L heat-resistant container, adding 50-200ml of pure water, and stirring in a water bath at 99 +/-1 ℃ to completely dissolve the agarose;
2) adding pure water to a constant volume of 1L, stirring and mixing uniformly, and keeping the temperature at 70 +/-5 ℃;
3) subpackaging a proper amount of the solution into a coplin jar, vertically immersing a clean glass slide into the completely melted agarose solution, standing for 1-30 seconds, lifting and immersing again to form a uniform film on the glass slide;
4) taking out the glass slide, wiping off agarose on the back surface by using absorbent paper, horizontally placing the glass slide on a smooth metal surface of a low-temperature (4 ℃) metal bath, and placing and cooling the glass slide to completely solidify the agarose; or the glass slide is horizontally placed at room temperature and naturally solidified;
5) the slide was placed horizontally in a drying oven or dry environment at a temperature range between room temperature and 100 ℃ until the agarose was completely dried and a fine film adhered to the glass was formed.
5. The sperm DNA fragment detection kit of claim 1, 2 or 3, wherein said low melting point gel is prepared by a method comprising:
1) weighing 1-50g of low-melting-point agarose, placing in a 1L heat-resistant container, adding 50-200ml of pure water, and stirring in a water bath at 99 +/-1 ℃ to completely dissolve the agarose;
2) adding pure water to a constant volume of 1L, stirring and mixing uniformly, and keeping the temperature at 45 +/-5 ℃;
3) the solution was portioned into 0.2ml sample tubes, 0.07ml per tube, while hot, to allow for natural coagulation.
6. The sperm DNA fragment detection kit of claim 1, 2 or 3, wherein the denaturant is prepared by a method comprising:
1) putting 200 plus 500mL of pure water into a 1L volumetric flask, adding 6.7-8.3mL of concentrated hydrochloric acid into the volumetric flask, and stirring to uniformly mix the pure water and the concentrated hydrochloric acid;
2) adding pure water to a constant volume of 1L, and stirring and mixing uniformly.
7. The sperm DNA fragment detection kit of claim 1, 2 or 3, wherein the lysate is prepared by the following method:
1) placing 800-900ml pure water into a 1L volumetric flask, weighing 117-146.3g of NaCl, 12.1-36.3g of Tris, 2.9-28.7g of Tris (2-carboxyethyl) phosphine hydrochloride and 0-5.8g of EDTA into the volumetric flask, and stirring to completely dissolve the materials;
2) weighing Triton X-1005-20 mL, adding into the volumetric flask, and stirring to completely dissolve;
3) slowly adding concentrated hydrochloric acid, and adjusting pH to 7.0-8.0;
4) the volume is adjusted to 1L by pure water.
8. The sperm DNA fragment detection kit of claim 1, 2 or 3 wherein said reishi stain is prepared by the following method:
1) weighing 0.25-2.5g of Swiss pigment and 0.05-0.5g of Gerstmann pigment, putting the weighed materials into a clean mortar, adding 5-20mL of methanol and 1-4mL of glycerol, grinding for a moment, and sucking out upper dye liquor; adding 5-20ml of methanol, continuously grinding, sucking the supernatant out, and repeating the steps for several times until the dye is completely dissolved;
2) the volume is adjusted to 500ml by methanol, the mixture is evenly stirred and transferred to a brown reagent bottle for storage.
9. The sperm DNA fragment detection kit of claim 1, 2 or 3, wherein said buffer is prepared by a method comprising:
1) putting 50-200ml of pure water into a 1L volumetric flask, weighing 0.57-5.7g of disodium hydrogen phosphate and 0.82-8.2g of monopotassium phosphate, adding into the volumetric flask, and stirring to completely dissolve the disodium hydrogen phosphate and the monopotassium phosphate;
2) detecting the pH value of the solution by using a pH meter, and adjusting the pH value to 6.0-7.0;
3) the volume is adjusted to 1L by pure water.
10. A sperm DNA fragment detection kit according to claim 1, 2 or 3, characterised in that said pre-coated slide is a slide coated with 1% aqueous agarose solution having a melting point of 20-60 ℃; the low-melting point gel is 1% of agarose hydrogel with the melting point of 20-65 ℃; the denaturant is an aqueous solution containing 0.08-0.1M HCl; the lysis solution is an aqueous solution containing 2-2.5M NaCl, 0.025-0.05M Tris (2-carboxyethyl) phosphine hydrochloride, 0.2M Tris, 1% Triton X-100, 0-10mM EDTA and pH 7.0-7.5; the Swiss stain solution is a glycerol-containing methanol solution containing 0.1 percent of Swiss pigment and 0.03 percent of Gerstmann pigment; the buffer solution is 0.06M phosphate buffer solution with the pH value of 6.4-6.8.
11. The method of detecting a sperm DNA fragment detection kit according to any one of claims 1 to 10 comprising the steps of:
(one) reagent preparation
1) Placing a sample tube pre-filled with 0.07ml of low-melting-point gel at 80-95 ℃ for incubation for 5-20 minutes, after complete melting, placing the sample tube at 37 ℃ for standby, and balancing for at least 5 minutes;
2) adjusting the room temperature to 20-25 ℃ before detection;
3) recovering the denatured liquid and the lysate to room temperature;
(II) sample preparation
1) Adjusting liquefied fresh sperm or thawed sperm frozen by liquid nitrogen or separated motile sperm to 5-10 × 10 with physiological saline6/ml；
(III) detection step
1) Taking 30 mu L of the sample to be detected with the concentration, adding the sample into the sample tube containing the melted low-melting-point gel, fully and uniformly mixing to prepare a sperm suspension, and incubating at 37 ℃ for later use;
2) placing the pre-coated glass slide in a refrigerator at 2-8 ℃ for precooling for 5 minutes, and then taking out, or horizontally placing the pre-coated glass slide on a metal bath precooled at 4 ℃, and quickly adding 5-30 mu L of the sperm suspension into a glass slide coated area;
3) cover the cover glass rapidly immediately, avoid producing the bubble; placing the mixture in a refrigerator at the temperature of 2-8 ℃ for 5-10 minutes to completely solidify the mixture; or standing in a pre-cooled metal bath at 4 deg.C for 5-10 min to solidify completely;
4) taking out the glass slide, and removing the cover glass along the horizontal direction;
5) the glass slide is immediately and accurately reacted with the denaturation liquid for 7 minutes;
6) taking out the glass slide, and sucking out liquid remained on the back of the glass slide by using filter paper; accurately reacting the glass slide with the lysis solution for 25 minutes;
7) taking out the glass slide, and sucking out liquid remained on the back of the glass slide by using filter paper; vertically immersing the glass slide in a large amount of pure water for 5 minutes;
8) taking out the glass slide, and sucking out liquid remained on the back of the glass slide by using filter paper; vertically immersing the glass slide into a reaction tank filled with 70% ethanol for 2 minutes;
9) taking out the glass slide, and sucking out liquid remained on the back surface of the glass slide by using filter paper; vertically immersing the glass slide into a reaction tank filled with 100% ethanol for 2 minutes;
10) naturally drying or air drying in the air;
11) covering each glass slide with about 150 mu L of Swiss stain, slowly adding about 300 mu L of buffer solution after 1-2min, lightly blowing and beating the mixed solution by an ear washing ball, standing at room temperature for 15min, and then lightly washing the stained slide with running water for about 3-6 s;
12) naturally drying or blow-drying;
13) 500 sperm were observed under a 40 × objective microscope, and the criteria for judgment were: fragmented sperm are obtained when the width of the halo is less than or equal to one third of the smallest diameter of the sperm head, and intact sperm are obtained when the width of the halo is greater than one third of the smallest diameter of the sperm head. And calculating the proportion of the number of the sperm fragmented from the DNA in the total number of the sperm to be detected, namely the sperm DNA fragmentation rate of the sample.
12. The method of detecting a sperm DNA fragment detection kit of claim 11, wherein: the reaction mode of the detection step 5) is that the glass slide is horizontally placed, 2mL of denatured liquid is immediately dripped into a sample gel area of the glass slide, the glass slide is inclined after reaction, and the filter paper is used for absorbing the liquid flowing down; and the detection step 6) adopts a reaction mode that the glass slide is horizontally placed, 2mL of lysate is dripped in a sample gel area, the glass slide is inclined after reaction, and the liquid flowing down is sucked by filter paper.
13. The method of detecting a sperm DNA fragment detection kit of claim 11, wherein: wherein the reaction mode of the detection step 5) is that the glass slide is immediately and vertically immersed into a reaction tank containing the denaturation liquid; and the detection step 6) adopts a reaction mode that the glass slide is vertically immersed into a reaction tank filled with a lysis solution.
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