Specific nucleic acid fluorescent staining reaction liquid and application thereof in sperm DNA integrity detection

A fluorescent staining and specific technology, applied in the field of cell biology, can solve the problems that cannot be confirmed with the naked eye or other means, cannot be found manually, and has cell debris, etc., and achieves convenient wavelength selection, good specificity, and operation steps. less effect

Pending Publication Date: 2022-04-12
重庆市人口和计划生育科学技术研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The advantage of this method is that it is detected by flow cytometry, which requires less labor and has high detection throughput, but the defects are also very obvious
First of all, the SCSA method uses fluorescence detection, and the detected results cannot be traced back, that is, the detected positive results cannot be confirmed with the naked eye or other means to confirm whether they are really positive; second, the semen of normal people is relatively clean, while in rare In the semen of men with weak sperm or inflammation, there are more impurities, such as cell debris, inorganic salts, etc.
These impurities can also electrostatically adsorb to acridine orange, resulting in a large number of false positives in flow cytometry, and these false positives cannot be detected manually

Method used

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  • Specific nucleic acid fluorescent staining reaction liquid and application thereof in sperm DNA integrity detection
  • Specific nucleic acid fluorescent staining reaction liquid and application thereof in sperm DNA integrity detection
  • Specific nucleic acid fluorescent staining reaction liquid and application thereof in sperm DNA integrity detection

Examples

Experimental program
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Effect test

Embodiment 1

[0078] Embodiment 1, a kind of specific nucleic acid fluorescent dyeing reaction liquid, comprises the following four: semen diluent (reagent A), acidification liquid (reagent B), fluorescent dye (reagent C), dye diluent (reagent D);

[0079] Semen diluent (reagent A): 0.01mol / L Tris hydrochloride, 0.15mol / L sodium chloride, 1mmol / L disodium edetate; the balance is water; adjust the pH to 7.3 ;

[0080] Acid treatment solution (reagent B): 0.08mol / L hydrochloric acid, 0.15mol / L sodium chloride, 0.1% (volume %) Triton X-100, and the balance is water;

[0081] Fluorescent dye (reagent C) is SGGV fluorescent dye, which is composed of SYBR GreenI and GoldView, which are mixed in a certain proportion before use;

[0082] Instructions: Dilute two 1000X dyes (SYBR GreenI and GoldView) with staining diluent to 2X working solution, then mix equal volumes and shake well to prepare SGGV fluorescent dye.

[0083] Staining diluent (reagent D): 40mmol / L Tris-acetic acid; 2mmol / L EDTA, the...

Embodiment 2

[0084] Embodiment 2. A method for detecting sperm DNA fragmentation, using the specific nucleic acid fluorescent staining reaction solution as described in Embodiment 1, and performing the following steps in sequence:

[0085] 1), preparation of fluorescent dyes:

[0086] Mix SYBR GreenI (1000X) and staining diluent at a volume ratio of 1:500 to obtain SYBR GreenI dilution (2X);

[0087] Mix GoldView (1000X) with staining diluent at a volume ratio of 1:500 to obtain GoldView diluent (2X);

[0088] Then, the SYBR GreenI dilution (2X) was mixed with the GoldView dilution (2X) in an equal volume ratio to obtain the SGGV fluorescent dye;

[0089] Note: The SGGV fluorescent dye prepared above can be stored in the dark for one week at 2-8°C.

[0090] 2), cell preparation:

[0091] For fresh semen samples, liquefy at room temperature for 30 minutes; for frozen semen samples, fully mix the thawed semen samples on ice (using an ice box); obtain the semen samples to be tested;

[00...

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Abstract

The invention discloses a specific nucleic acid fluorescent staining reaction solution which comprises the following four components: a semen diluent, an acidizing fluid, a fluorescent dye and a dye diluent. The invention also provides a method for detecting sperm DNA fragmentation by using the specific nucleic acid fluorescent staining reaction liquid. The method comprises the following steps: preparing a fluorescent dye; preparing cells; cracking and dyeing the cells; detecting on a machine; result judgment: determining that the spermatids emitting green fluorescence are normal spermatids according to a photographing result; the cells emitting orange and red fluorescence are fragmented sperm cells. Furthermore, the DNA damage proportion in the sample can be calculated according to the number ratio of the two fluorescent cells. The method provided by the invention has the advantages of few operation steps, good specificity and accurate judgment on sperm DNA fragments.

Description

technical field [0001] The invention belongs to the field of cell biology, in particular to a fluorescent reaction solution for sperm DNA fragmentation detection and its application in sperm DNA integrity detection. Background technique [0002] Sperm DNA is the carrier of sperm genetic material. Recent studies have shown that the integrity of sperm DNA can affect the fertilization ability of sperm, the division of fertilized eggs and the development of embryos. Sperm DNA integrity is critical for natural conception, artificial insemination, embryo, fetus, infant and even adult development. Studies have shown that sperm DNA integrity is an independent parameter superior to routine semen analysis and is associated with assisted reproductive technology outcomes. Therefore, the analysis of sperm DNA integrity in male infertile patients is helpful to explore or discover the causes of infertility, and provide consultation and diagnosis basis for the prevention and treatment of p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N15/14G01N1/38G01N1/30G01N1/28
Inventor 焦爽丽张益岳平杨继高叶鑫洪旭涛
Owner 重庆市人口和计划生育科学技术研究院
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