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Sperm DNA fragment detection kit and detection method thereof

A technology for detecting kits and sperm, applied in the preparation of test samples, measuring devices, sampling, etc., can solve the problems of reagent irritating odor, affecting cracking efficiency, loss of reducibility, etc., to reduce health hazards and improve working environment , The effect of reducing material cost

Pending Publication Date: 2022-02-25
BEIJING INST OF HUMAN GENETICS & REPRODUCTION MEDICINE LTD CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

2) Dithiothreitol is easily oxidized by oxygen in the air. In practical applications, as the user repeatedly uses it and continuously contacts the air, it will gradually lose its reducibility and weaken the cracking effect of the lysate.
The acidic hydrochloric acid solution that provides DNA denaturation conditions is often partly brought into the subsequent lysate along with the sample slide, reducing the pH of the lysate and affecting the reducibility of dithiothreitol in it.
4) As an expensive compound, the high concentration of dithiothreitol also makes the cost of detection reagents relatively high
However, the lower concentration of dithiothreitol in this method also affects the cracking efficiency accordingly, making the halo not obvious, which affects the accuracy of interpretation of the results
Sodium dodecyl sulfate (SDS) easily damages the tail of sperm and also affects the identification of cell types
[0007] Second, the technical problems brought about by the relatively fixed process framework
There are generally the above-mentioned technical problems to be solved, including the presence of obvious pungent odor, instability, and high cost in all reagents.

Method used

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  • Sperm DNA fragment detection kit and detection method thereof

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Embodiment 1

[0089] This embodiment provides a sperm DNA fragment detection kit, including: pre-coated glass slide, low melting point gel, denaturing solution, lysate, Wright's stain and buffer;

[0090] The pre-coated glass slide is a glass slide coated with 1% agarose aqueous solution with a melting point of 20-60°C; the low melting point gel is 1% agarose hydrogel with a melting point of 20-65°C gel; the denaturing solution is an aqueous solution containing 0.1M HCl; the lysate contains 2.5M NaCl, 10mM tris (2-carboxyethyl) phosphine hydrochloride, 0.2M Tris, 1% Triton X-100, 5mM EDTA, pH is the aqueous solution of 7.0-7.5; The Wright's staining solution is the glycerol-containing methanol solution containing 0.1% Wright's pigment and 0.03% Gibson's pigment; The Wright's buffer is 0.06M of pH6.4-6.8 Phosphate buffer.

[0091] The preparation method of described pre-coated glass slide is:

[0092] 1) Weigh 10g of low-melting point agarose and place it in a 1L heat-resistant container, ...

Embodiment 2

[0117] This embodiment provides a sperm DNA fragment detection kit, including: pre-coated glass slide, low melting point gel, denaturing solution, lysate, Wright's stain and buffer;

[0118] The pre-coated glass slide is a glass slide coated with 1% agarose aqueous solution with a melting point of 20-60°C; the low melting point gel is 1% agarose hydrogel with a melting point of 20-65°C glue; the denaturing solution is an aqueous solution containing 0.1M HCl; the lysate contains 2.5M NaCl, 50mM tris (2-carboxyethyl) phosphine hydrochloride, 0.2M Tris, 1% Triton X-100, 5mM EDTA, pH is the aqueous solution of 7.0-7.5; The Wright's staining solution is the glycerol-containing methanol solution containing 0.1% Wright's pigment and 0.03% Gibson's pigment; The Wright's buffer is 0.06M of pH6.4-6.8 Phosphate buffer.

[0119] The preparation method of described lysate is:

[0120] 1) Put 900ml of pure water in a 1L volumetric flask, weigh 146.3g of NaCl, 24.2g of Tris, 14.3g of tris(...

Embodiment 3

[0126] This embodiment provides a sperm DNA fragment detection kit, including: pre-coated glass slide, low melting point gel, denaturing solution, lysate, Wright's stain and buffer;

[0127] The pre-coated glass slide is a glass slide coated with 1% agarose aqueous solution with a melting point of 20-60°C; the low melting point gel is 1% agarose hydrogel with a melting point of 20-65°C gel; the denaturing solution is an aqueous solution containing 0.1M HCl; the lysate contains 2.5M NaCl, 100mM tris (2-carboxyethyl) phosphine hydrochloride, 0.2M Tris, 1% Triton X-100, pH It is an aqueous solution of 7.0-7.5; the Wright's stain is a glycerin methanol solution containing 0.1% Wright's pigment and 0.03% Gibson's pigment; the Wright's buffer is 0.06M phosphate buffered saline with a pH of 6.4-6.8 liquid.

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Abstract

A sperm DNA fragment detection kit is characterized by comprising a pre-coated glass slide, low-melting-point gel, a denaturation solution, a lysis solution, a Wright's staining solution and a buffer, and is characterized in that the lysis solution contains a reducing agent tris(2-carboxyethyl) phosphine hydrochloride. The lysis solution contains tris(2-carboxyethyl) phosphine hydrochloride as a reducing agent, disulfide bonds contained in protamine packaging sperm DNA can be efficiently reduced and opened, no pungent smell is generated, potential health hazards to a user are reduced, use of equipment such as an exhaust hood is omitted, and the working environment of the user is improved. As the use concentration of the tris(2-carboxyethyl) phosphine hydrochloride is remarkably low, remarkable economic benefits of greatly reducing the cost of production and used materials are brought.

Description

technical field [0001] The invention belongs to in vitro detection reagents, in particular to a sperm DNA fragment detection kit and a detection method thereof. Background technique [0002] The degree of sperm DNA fragmentation is an important indicator reflecting the integrity of the genetic material carried by male sperm. Abnormal DNA fragmentation in male sperm is a common factor leading to male infertility. Currently widely used detection methods mainly include sperm chromatin structure analysis (SCSA) and sperm chromatin diffusion (SCD). The sperm chromatin diffusion assay is an in situ assay performed on glass slides. The detection principle is that spermatozoa with more DNA double-strand breaks due to congenital or acquired factors are prone to unzipping at the double-strand break site to produce single-stranded DNA after being treated with DNA denaturing solution, and then combined with the nuclear matrix combine with each other. Under high salt and reducing con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N1/30G01N21/84
CPCG01N1/2813G01N1/30G01N21/84
Inventor 唐军李存玺马雁南
Owner BEIJING INST OF HUMAN GENETICS & REPRODUCTION MEDICINE LTD CHINA
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