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Method and kit for detecting sperm DNA fragment rate

A fragmentation rate and kit technology, applied in the field of in vitro diagnostic reagents, can solve problems such as complex operation process, incomplete enzymatic hydrolysis, complex methods, etc., and achieve accurate and repeatable results

Pending Publication Date: 2020-06-19
ZHEJIANG CELLPRO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] (2) Sperm chromatin dispersion (SCD): It was the standard method for detecting DFI before the invention of flow cytometry. The operation process is complicated, takes a long time, and relies on subjective judgment
[0008] (3) Comet test: Quantitative detection, counting 200-500 sperm by manual observation each time, long detection time, complex method, strong subjectivity, and difficult standardization
This method has some disadvantages, such as incomplete enzymatic digestion of DNA, which can lead to a higher detection value than the true value
[0011] (6) Fluorescent in situ labeling method: direct, objective, efficient and fast, but the application is narrow, mostly used for chromosome euploid detection
However, when the sperm DNA is detected by traditional flow cytometry, the RNA in the sperm will also be in a single-stranded state, which is also combined with acridine orange, and the emitted fluorescence is the same as that emitted by the combination of acridine orange / single-stranded DNA, resulting in The DNA detection signal includes the RNA detection signal, and such detection results cannot fully, accurately and truly reflect the status of the sperm DNA itself

Method used

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  • Method and kit for detecting sperm DNA fragment rate
  • Method and kit for detecting sperm DNA fragment rate
  • Method and kit for detecting sperm DNA fragment rate

Examples

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Effect test

Embodiment 1

[0056] The present embodiment provides the use of the method of the present invention to detect sperm DNA fragmentation rate, wherein the detection of RNase enzyme digestion effect comprises the following steps:

[0057] 1.1 Preparation of items:

[0058] Prepare sterile, disposable plastic products (centrifuge tube, tip), Trizol reagent, chloroform, isopropanol, 75% ethanol, RNase-free water, RNase A, nucleic acid quantitative detector.

[0059] 1.2 Extraction and isolation of total RNA from sperm cells:

[0060] 1.2.1 Lysis of sperm cells (cell volume 5×10 8 indivual )

[0061] The liquefied semen was centrifuged at 800g for 10 minutes at room temperature, and the supernatant was discarded completely. Add 1mL Trizol and immediately mix by pipetting 5 times.

[0062] 1.2.2 Let stand at room temperature for 5 minutes. Then add 0.25mL of chloroform, and vigorously invert the centrifuge tube to mix. After standing for 5 minutes, centrifuge at a centrifugal speed of 12000g ...

Embodiment 2

[0077] This example provides the detection of sperm DNA fragmentation rate using different methods.

[0078] Specifically, the following steps are included:

[0079] 2. Sample Preparation

[0080] 2.1 Divide 20 naturally liquefied semen (each sample volume is 1.5-5.5mL, sperm concentration is 15-200 million / mL) into 3 parts, and use the following 3 methods to detect sperm DFI: method A: flow cytometry of digested RNA Cytometry to detect DFI; method B: flow cytometry to detect DFI after undigested RNA; method C: sperm chromatin diffusion method to detect DFI;

[0081] 2.2 Method A detects DFI:

[0082] 2.2.1 Dilute sperm with 37°C buffer to 1-2×10 6 pcs / mL; the buffer solution is a mixture of 0.15 mol / L NaCl (sodium chloride), 1 mmol / L EDTA (ethylenediaminetetraacetic acid), pH 7.4;

[0083] 2.2.2 Add 500 μL of diluted sperm into the sample tube of the flow cytometer, add 5 μL of 10 mg / mL RNase A (purchased from Sigma), and digest at 37°C for 5 minutes;

[0084] After 2.2....

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Abstract

The invention provides a method and a kit for detecting sperm DNA fragment rate. According to the method and the kit provided by the invention, RNA in a sperm sample is digested by RNA enzyme based onflow cytometry, so that mixed signals brought by sperm RNA are eliminated, the detection result is more accurate, stable and reliable, the repeatability is good, and the clinical popularization is easy.

Description

technical field [0001] The application belongs to the field of in vitro diagnostic reagents, in particular to a method and a kit for detecting sperm DNA fragmentation rate. Background technique [0002] 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 phosphate molecules are connected by ester bonds to form a long-chain DNA skeleton, which is arranged on the outside, and four bases are arranged on the inside, following the principle of complementary base pairing. Each sugar molecule is connected to one of the four bases. The sequence of these bases arranged along the long DNA chain can constitute the genetic code and guide the synthesis of proteins. [0003] The main function of sperm is to retain and pass on the genetic information of the parents. Each normal sperm contains 23 chrom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N15/14G01N21/64
CPCG01N15/14G01N21/6428
Inventor 曾桥胡西陵陈继勇
Owner ZHEJIANG CELLPRO BIOTECH
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