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Sperm DFI (DNA Fragmentation Index) detection method based on flow cytometry

A flow cytometry and detection method technology, applied in the field of sperm DFI detection based on flow cytometry, can solve the problems of low detection efficiency, strong subjectivity of detection results, poor repeatability, etc., and achieve high-throughput detection and calculation Effect

Inactive Publication Date: 2017-07-07
ZHEJIANG CELLPRO BIOTECH
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Problems solved by technology

The company processes and stains the sperm samples through the DFI kit, and then automatically analyzes the samples by flow cytometry. The detection results of this method are too dependent on human operation, and the detection results are highly subjective; Poor sex, poor sensitivity characteristics

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  • Sperm DFI (DNA Fragmentation Index) detection method based on flow cytometry
  • Sperm DFI (DNA Fragmentation Index) detection method based on flow cytometry

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[0018] A method for detecting sperm DFI based on flow cytometry, comprising the following steps:

[0019] (1) Dilute the liquefied semen with 37°C buffer to 1~2×10 6 each / mL; the buffer solution is sodium chloride, potassium dihydrogen phosphate, glucose, potassium chloride, magnesium chloride, calcium chloride, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid disodium salt , a mixture of sodium bicarbonate, sodium pyruvate, bovine serum albumin and sodium lactate;

[0020] (2) Add 500 μL of diluted sperm into the sample tube of the flow cytometer, and add acidification treatment buffer (0.08mol / L hydrochloric acid, the purpose is to make the compact DNA looser, so as to facilitate the subsequent staining steps carry out), add AO staining solution after 30 seconds; after the acidification treatment, use the characteristics of acridine orange dye combined with double-stranded DNA to emit green or yellow fluorescence, and combined with single-stranded DNA to emit red fluoresc...

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Abstract

The invention discloses a sperm DFI (DNA Fragmentation Index) detection method based on flow cytometry. The sperm DFI detection method is characterized by comprising the following steps: (1) diluting liquefied seminal fluid to 1-2*10<6> / mL by a buffer solution at the temperature of 37DEG C; (2) adding 500mu L of diluted sperms into a flow cytometer sample loading tube, adding an acidizing buffer solution, and adding an AO (Acridine Orange) staining solution after 30s; (3) setting the calibration of a flow cytometer, putting a detection tube on a machine, and measuring each tube sample for at least two continuous times, wherein each tube sample at least records 5000 cells and carries out statistical analysis by DFIView software; (4) judging ways to carry out result judgment; (5) after measurement is carried out, respectively using decolorizer, sample feeding tube detergent and degerming double distilled water to thoroughly remove cell debris and fluorescent dye in a sample feeding line of the flow cytometer. The sperm DFI detection method has the advantages of simpleness in operation, accurate detection result, stability, reliability, good repeatability and easiness in clinical popularization.

Description

technical field [0001] The invention relates to the field of in vitro diagnostic reagents, in particular to a method for detecting sperm DFI based on flow cytometry. Background technique [0002] Traditional semen testing methods mainly focus on sperm DNA fragmentation, and these test results cannot fully and accurately reflect the status of sperm. The main function of sperm is to retain and pass on the genetic information of the parents, while the DNA exists in the sperm and is wrapped in a dense structure by protamine. DNA is a long-chain polymer composed of four deoxynucleotides, namely: adenine deoxynucleotide (dAMP), thymidine deoxynucleotide (dTMP), cytosine deoxynucleotide (dCMP) , Guanine deoxynucleotide (dGMP). Deoxyribose (five-carbon sugar) and phosphoric acid molecules are connected by ester bonds to form a long-chain skeleton, arranged on the outside, and four bases are arranged on the inside, following the principle of complementary base pairing. Each sugar ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N15/14
CPCG01N15/14
Inventor 曾桥江南房海燕陈继勇胡润环
Owner ZHEJIANG CELLPRO BIOTECH
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