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Detecting agent for detecting male infertility and use thereof

A technology of male infertility and detection reagents, which is applied in the direction of microbial measurement/inspection, fluorescence/phosphorescence, biochemical equipment and methods, etc., which can solve the uncertain factors that increase technical complexity, large investment in disposable equipment, and easy pollution and other issues to achieve specific coverage, great clinical significance, and eliminate false negative effects

Inactive Publication Date: 2011-12-14
BEIJING GP MEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, there is an electrophoresis detection process after PCR amplification, which is cumbersome to operate and easy to cause pollution, which increases the technical complexity and a large number of uncertain factors to realize the detection
Although Real-Time PCR is more sensitive than the former and does not require electrophoresis detection, the current application of this technology is the same as the STS-PCR method, and the user's disposable equipment investment is large

Method used

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  • Detecting agent for detecting male infertility and use thereof
  • Detecting agent for detecting male infertility and use thereof
  • Detecting agent for detecting male infertility and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0143] Example 1: Preparation of GLP DAZ a / GLP DAZ b / CSP 18 probe set

[0144] The DAZ probe was prepared using the random priming method, and the template DNA was labeled as fluorescent red (Tetramthylrhodamine-5-dUTP, purchased from Roche). The probe preparation kit was purchased from Roche (Random Primed DNA Labeling Kit)

[0145] 1. Template DNA preparation (take RP11-263A15 as an example):

[0146] (1) Take an appropriate amount of purchased clone RP11-263A15 (existing in the form of bacterial liquid) for amplification culture;

[0147] (2) Pick a single colony grown on LB solid medium, inoculate it in 20ml LB (containing Amp100μg / ml) liquid medium, culture at 37°C and shake at 250rmp overnight (about 12-14 hours);

[0148] (3) Pour 1.5ml of culture solution into a 1.5ml eppendorf tube and centrifuge at 12000rmp for 1-2 minutes. Discard the supernatant, and place the centrifuge tube upside down on toilet paper for a few minutes to drain the liquid as much as possible; ...

Embodiment 2

[0183] Example 2: Preparation of peripheral blood chromosomes before FISH operation

[0184] 1. Reagents:

[0185] Colchicine, medium, hypotonic solution, glacial acetic acid, 100% ethanol.

[0186] 2. Experimental operation

[0187] (1) Take fresh blood within 72 hours, immediately put the injection needle directly through the rubber stopper of the culture bottle, inject 30-40 drops of whole blood into the culture medium, shake it gently, and then culture it in a 37°C incubator;

[0188] (2) Cultivation: the time is 70±2 hours. During the culture period, shake gently regularly to make the cells fully contact with the culture medium;

[0189] (3) 2-4 hours before terminating the culture, add colchicine to the culture medium (add four drops with a 5ml syringe to make the final concentration 0.07-0.08 μg / ml);

[0190] (4) Transfer the culture completely into a clean centrifuge tube, centrifuge at 1000rpm for 8 minutes, and discard the supernatant;

[0191] (5) Add 5ml of pr...

Embodiment 3

[0199] Embodiment 3: the operation of fluorescence in situ hybridization (FISH)

[0200] The FISH operation mainly includes three steps: denaturing hybridization, slide washing, and counterstaining. Among them, there are two methods of denaturing hybridization: formamide denaturing hybridization and hybridization instrument denaturing hybridization. The former is to denature the samples and probes separately, and the latter is to co-denature the samples and probes with a hybridization instrument, which reduces the influence of human factors.

[0201] 1. Denaturation hybridization

[0202] 1. Formamide denaturation hybridization (manual operation)

[0203] (1) Probe mixture preparation and denaturation

[0204] Probe mix preparation. Add the following samples to microcentrifuge tubes at room temperature:

[0205] Table 8 Probe Mixture Preparation

[0206] name Volume (μl) GLP hybridization buffer 7.0 Deionized water 1.0 Three probe sets pr...

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Abstract

The invention relates to a detection reagent and a kit for detecting male-sterility. Concretely, the detection reagent comprises 1) at least one of a GLP DAZ a probe set and a GLP DAZ b probe set, and 2) a CSP 18 probe set. The kit comprises the detection reagent dissolved in a TE Buffer, wherein each one of the probe sets in the kit has concentration of 50 to 300 ng / microliter. The invention also relates to a use of the detection reagent and the kit in detection of DAZ gene family deletion, and a method for detection of DAZ gene family deletion.

Description

technical field [0001] The invention relates to a detection agent and a kit for detecting male infertility, the use of the detection agent and the kit in detecting the deletion of the DAZ gene family, and a method for detecting the deletion of the DAZ gene family. Background technique [0002] In recent years, the number of infertility patients has increased day by day. The World Health Organization predicts that in the 21st century, infertility will become the third largest disease after tumors and cardiovascular and cerebrovascular diseases. According to statistics, there are 60 million to 80 million couples in the world with infertility, and about 10% to 15% of couples in my country are infertile, of which male factors account for 40% to 50%. higher than in rural areas. [0003] Small deletions on the Y chromosome can cause spermatogenesis disorders, leading to azoospermia or severe oligospermia. Studies have found that there is a region related to sperm production in t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 陈忠程晓蕾成华吴晓东
Owner BEIJING GP MEDICAL TECH
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