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Sperm DNA fragmentation fluorescence detection method, detection kit and application thereof

A fluorescent detection and fragmentation technology, applied in the field of sperm quantitative detection and analysis, can solve the problems of lack of comprehensive and objective evaluation of sperm quality and reproductive outcomes, and the inability to directly quantitatively detect the degree of sperm DNA fragmentation, and achieve simple and fast operation , good clinical application ability, time-consuming effect

Pending Publication Date: 2021-06-29
HUAZHONG UNIV OF SCI & TECH
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Problems solved by technology

At present, 15% and 30% are usually used as the laboratory reference value of DFI in clinical practice. According to DFI30%, the sperm DNA integrity is qualitatively divided into good, good, Fair, poor, and due to the lack of standard values ​​for SDF detection, qualitative analysis by setting DFI thresholds cannot comprehensively and objectively evaluate sperm quality and reproductive outcomes
[0006] To sum up, none of the existing methods can directly quantitatively detect the fragmentation degree of sperm DNA, which is a big gap in the field of reproductive clinical detection

Method used

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  • Sperm DNA fragmentation fluorescence detection method, detection kit and application thereof
  • Sperm DNA fragmentation fluorescence detection method, detection kit and application thereof
  • Sperm DNA fragmentation fluorescence detection method, detection kit and application thereof

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Embodiment 1

[0036] The fluorescence detection method for the number of sperm DNA breakpoints provided in this embodiment of the present invention includes four steps: extraction of sperm genome DNA, extension of sperm DNA breakpoints, fluorescent probe detection, and data processing. Specifically as follows:

[0037] 1. Extraction of Genomic DNA from Sperm

[0038] According to the extraction method of Tiangen Cell Genome Extraction Kit, the DNA extraction was carried out. In order to further improve the extraction purity and extraction amount of sperm DNA, some links in the extraction process were optimized:

[0039] (1) Centrifuge 200 μL of sperm at 12,000 rpm for 1 minute, add 200 μL of Solution A and mix well. Mix well, centrifuge at 12000rpm for 1 minute.

[0040] (2) Discard the supernatant and repeat the above steps once.

[0041] (3) Discard the supernatant, take about 50 μL of Solution A from the bottom of the EP tube, and pipette it evenly.

[0042] (4) Add 250 μL Solution B...

Embodiment 2

[0069] In this embodiment, human semen samples with different sperm qualities are tested for sperm DNA, including the following basic steps:

[0070](1) 13 human semen samples collected by masturbation were collected. Routine semen analysis showed that 8 of them were normal sperm samples and 5 were weak sperm samples. Normal sperm, that is, the specimen also meets the semen volume ≥ 1.5ml, sperm concentration ≥ 15×106 / mL, sperm forward movement ≥ 32%, and sperm motility ≥ 40% stipulated in the "World Health Organization Human Semen Inspection and Processing Laboratory Manual" , the parameter standard, and the DFI of the normal samples taken in this test is less than or equal to 30%. Asthenic sperm sample: semen volume ≥ 1.5ml, sperm concentration ≥ 15×106 / mL, sperm forward movement 30%.

[0071] (2) Tiangen Sperm DNA Extraction Kit was used to extract sperm genomic DNA. For each specimen, sterile deionized water was used to adjust the concentration of sperm DNA to 20 ng / μL. ...

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Abstract

The invention belongs to the technical field of sperm quantitative detection and analysis and particularly relates to a sperm DNA fragmentation number fluorescence detection method, a detection kit and application thereof. Based on defects of an existing SDF detection method and incomplete evaluation index DFI, the application aims at the current situation that an accurate quantitative evaluation for sperm DNA fragmentation degree, which can directly measure DNA damage, is convenient and rapid to realize and has simple requirements, is lacked at present, and aims to meet national requirements. According to the invention, the number of sperm DNA breaking sites is creatively used as an index for evaluating the integrity of sperm DNA and introduced into a PCR detection platform, and an enzyme-assisted sperm DNA breaking point number fluorescence detection kit and a detection method are developed.

Description

technical field [0001] The invention belongs to the technical field of sperm quantitative detection and analysis, and in particular relates to a fluorescent detection method for sperm DNA fragmentation number, a detection kit and an application thereof. Background technique [0002] Affected by multiple factors such as aging, lifestyle, environment and work pressure, the proportion of infertility patients in my country is increasing year by year, and the incidence rate is about 10% to 15%, of which the male factor accounts for about 40%. At the same time, the quality of men's semen has been declining year by year. With the increasing incidence of infertility, it is more and more important and urgent to evaluate the semen quality and fertility of men accurately and rationally. [0003] At present, the indicators of routine semen analysis mainly focus on evaluating the general properties of semen (semen volume, color and transparency, odor, etc.) semen quality. However, rou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64C12N15/10
CPCG01N21/6486C12N15/1003C12N15/10
Inventor 肖先金吴曈勃颜贝叶玮聪
Owner HUAZHONG UNIV OF SCI & TECH
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