Method for detecting spermatozoon DNA fragment with spermatozoon chromatin diffusion experiment

A diffusion experiment and chromatin technology, which is applied in the field of sperm chromatin diffusion experiment to detect sperm DNA fragments, can solve the problems of odor, irritation and corrosion, expensive kits, environmental pollution, etc., to reduce pollution and low cost , high resolution effect

Inactive Publication Date: 2008-12-10
SHANDONG PROVINCIAL FAMILY PLANNING INST OF SCI & TECH
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Problems solved by technology

The SCD detection method has many advantages, such as: simple, fast, accurate, and high resolution. However, because the kit contains intellectual property rights, the kit is expensive. Due to this limitation, it has not been widely used in my country at present, and this The sperm nucleoprotein ...

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  • Method for detecting spermatozoon DNA fragment with spermatozoon chromatin diffusion experiment
  • Method for detecting spermatozoon DNA fragment with spermatozoon chromatin diffusion experiment
  • Method for detecting spermatozoon DNA fragment with spermatozoon chromatin diffusion experiment

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Embodiment Construction

[0024] A method for detecting sperm DNA fragments in a sperm chromatin diffusion experiment according to the present invention, the steps are as follows:

[0025] ① Precise preparation of sperm lysate

[0026] The sperm lysate contains 4.844 grams of trishydroxymethylaminomethane, 11.7 grams of sodium chloride, 1.86 grams of dithiothreitol, 8.12 grams of saponin or bellflower extract and disodium edetate per 100 ml of distilled water 12.32 grams, pH 7.5;

[0027] ② Store the lysate at 4°C for 2 weeks, take it out 1.5 hours before use, and place it in a room temperature of 22°C;

[0028] ③ Dilute the semen to 5-10×10 6 / ml, dilute 0.3ml of semen at room temperature 37°C, add 0.7ml of 1% low-melting point agarose, and mix well;

[0029] ④ Take 50 microliters of diluted semen and place it on a glass slide treated with 0.65% agarose, cover it with a 22×22mm cover glass, and place it in the refrigerator for 5 minutes, and the temperature in the refrigerator is 4°C;

[0030] ⑤Re...

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Abstract

The invention discloses a method for detecting sperm DNA fragmentations by a sperm chromatin dispersion test, which comprises the following steps that: 1) a sperm lysis solution is accurately prepared; 2) the sperm lysis solution is placed at a temperature of 4 DEG C and is preserved for 2 weeks, is taken out 1.5 hours earlier before the use, and is placed at a room temperature of 22 DEG C; 3) semen is diluted to 5-10x10<6>/ml; 4) 50 microlitres of diluted semen is taken and placed in a refrigerator for 5 minutes, and the temperature in the refrigerator is 4 DEG C; 5) a microscopic glass is removed after the diluted semen is taken out from the refrigerator, and a glass slide is horizontally immersed into hydrochloric acid for degradation for 7 minutes, and then is taken out; 6) the glass slide taken away in the step 5) is horizontally immersed into the sperm lysis solution in the step 1) for 20 minutes; 7). The glass slide in the step 6) is washed by a buffer liquid for 5 minutes; 8) alcohol is used to respectively dehydrate for 2 minutes; and 9) a Wright method or a Giemsa-Wright method or a sky blue method is used to dye sperms, and the sperm DNA fragmentations are observed and analyzed under an optical microscope. The method realizes the aims of simplicity, quickness, accuracy, low cost, high resolution of sperm DNA detection.

Description

technical field [0001] The invention relates to reagents used in clinical medicine, and is a method for detecting sperm DNA fragments in a sperm chromatin diffusion experiment. Background technique [0002] At present, many countries in the world are studying the abnormality of DNA integrity caused by the formation of DNA fragments due to the influence of internal and external environments during sperm maturation, resulting in abnormal sperm morphology and function. Since the integrity of DNA in male infertility testing is a routine test of semen and the main indicator for evaluating male infertility, how to detect sperm DNA integrity is one of the important topics in the field of reproductive medicine. The leading detection method in foreign countries is the sperm chromatin diffusion test, its English name is spermchromatin dispersion, referred to as SCD, and its commercial test kit has been widely used. The SCD detection method has many advantages, such as: simple, fast, ...

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Application Information

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IPC IPC(8): C12Q1/68G01N1/30
Inventor 邱毅王磊光张丽红
Owner SHANDONG PROVINCIAL FAMILY PLANNING INST OF SCI & TECH
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