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Atp6 expression level as a kit for assessing patient fertility and its application

A technology of expression level and fertility, applied in the field of biomedicine, can solve problems such as unreported, achieve the effect of good application prospect, convenient sampling and easy detection

Active Publication Date: 2021-06-15
SHANGHAI FIRST PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports about the ATP6 expression level of the present invention as a test kit for evaluating patient fertility and its application

Method used

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  • Atp6 expression level as a kit for assessing patient fertility and its application
  • Atp6 expression level as a kit for assessing patient fertility and its application
  • Atp6 expression level as a kit for assessing patient fertility and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 The preparation method of the diagnostic kit that detects patient's fertility disease

[0033] The production process and operation flow of the diagnostic kits for detecting patients' fertility diseases are mainly based on PCR technology.

[0034]A type of sperm mRNA related to patients with abnormal fertility was screened by quantitative PCR and other technologies, as an indicator for predicting whether there are normal semen parameters and abnormal fertility diseases.

[0035] This kit includes a batch of primers, the primer sequences are as follows:

[0036]

[0037] Use the SYbrGreen method for fluorescence real-time quantitative PCR detection, and use the collected CT value to calculate the relative ACTB amount of ATP6. The calculation formula is: m ATP6 / m ACTB =2^(CT ACTB -CT ATP6 ), if the calculation result is ATP6 / ACTB<5, it indicates that the subject has a potential risk of infertility even if the relevant semen parameters are normal.

Embodiment 2

[0038] Example 2 The method of using the diagnostic kit for detecting the patient's fertility disease

[0039] The method of use of the diagnostic kit for detection of fertility disorders in patients is as follows:

[0040] (1) Purify sperm by gradient density centrifugation: take 0.5 mL of 90% gradient solution and add it to a 5 mL round-bottomed centrifuge tube, cover with 0.5 mL of 45% gradient solution, take 1 mL of liquefied semen and carefully cover the gradient solution, Centrifuge at 600G for 20-30min, carefully suck off the semen layer and 45% gradient liquid layer, remove the cells at the junction of the layers, add one volume of HTF solution containing 5% HSA to wash the sperm settled in the 90% layer, and remove after centrifuging at 600G for 20min Supernatant, precipitate was lysed with 500uL Trizol reagent;

[0041] (2) Use the Trizol method or the spin column method to carry out sperm RNA extraction: the Trizol method is: after the semen is purified, centrifuge...

Embodiment 3

[0046] Embodiment 3 clinical verification

[0047] The experiments in this example were all carried out on the premise of the informed consent of the person himself, and he signed the informed consent form.

[0048] 1 Experimental data

[0049] The semen of adult men with normal parameters and normal fertility diagnosed by the hospital and the semen of adult male patients with normal parameters but birth defects were collected and divided into normal group and patient group respectively.

[0050] 2 Experimental methods

[0051] Sperm from the normal group and the patient group were processed as follows:

[0052] (1) Purify sperm by gradient density centrifugation: take 0.5 mL of 90% gradient solution and add it to a 5 mL round-bottomed centrifuge tube, cover with 0.5 mL of 45% gradient solution, take 1 mL of liquefied semen and carefully cover the gradient solution, Centrifuge at 600G for 20-30min, carefully suck off the semen layer and 45% gradient liquid layer, remove the...

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Abstract

The present invention relates to the application of ATP6 expression level as a patient's fertility assessment and its kit. The present invention first proves that the ATP6 expression level can be used as a reference index for evaluating the fertility of patients with normal semen parameters. By purifying semen, extracting sperm RNA for sequencing, cDNA Synthesis, using the SYbrGreen method for fluorescence real-time quantitative PCR detection, complete the detection of ATP6 expression level, and diagnose patients with normal semen parameters and abnormal fertility. The advantages are as follows: (1) The present invention provides a new application for the expression level of ATP6. (2) Sampling is convenient and easy to detect. (3) The invention can provide a theoretical basis for the treatment of abnormal fertility, and has a good application prospect.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to the expression level of ATP6 as a kit for evaluating the fertility of patients and its application. Background technique [0002] Nearly 15% of couples fail their first pregnancy attempts, and data from the past 20 years show that approximately 30% of these clinical cases occur in males alone and another 20% occur in couples with abnormalities. Thus, men are responsible for at least half of infertile couples in approximately 50%. Male infertility is an important medical problem that affects most people. The cause of male infertility in about half of all cases often does not reveal any specific cause, even after a thorough examination. At present, the research on these factors mostly stays at the single gene level and genome level. Studies have shown that male infertility is not an independent disease, but the result of a combination of multiple diseases or multiple factors...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/158
Inventor 朱子珏李铮乔中东王升跃徐汪节李冲
Owner SHANGHAI FIRST PEOPLES HOSPITAL