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Multiplex-PCR detection method for single sperm

A detection method and single sperm technology, applied in the field of PCR detection, to achieve the effect of wide application, high efficiency and wide clinical application

Inactive Publication Date: 2016-03-23
REPRODUCTIVE & GENETIC HOSPITAL OF CITIC XIANGYA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, the detection of reproduction is mainly focused on women's pre-pregnancy, prenatal and newborns. Sperm is the only haploid that exists in men, and the detection of male germ cells is still in the exploratory stage.

Method used

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  • Multiplex-PCR detection method for single sperm
  • Multiplex-PCR detection method for single sperm
  • Multiplex-PCR detection method for single sperm

Examples

Experimental program
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Effect test

Embodiment 1

[0041] A single sperm multiple PCR detection method, comprising the following steps:

[0042] A. Sperm pretreatment (Using the swimming ability of motile sperm, that is, it can swim through the liquid interface and enter different culture fluids, so as to eliminate dead sperm, poorly motile sperm, agglutinated sperm, deformed sperm, red and white blood cells and other harmful components. and impurities are separated by themselves):

[0043] (A1) Divide the liquefied semen to be tested evenly into two centrifuge tubes, and then add culture medium F-10 (supplier: vitrolife) twice the volume of the semen to be tested;

[0044] (A2) Pipette evenly, mix well, and centrifuge at 1200r / min for 10min;

[0045] (A3) Discard the supernatant, then add 2ml of culture medium F-10, mix the sperm sediment thoroughly, and centrifuge at 1200r / min for 10min;

[0046] (A4) Discard the supernatant, keep about 0.3ml, gently break up the sperm sediment at the bottom of the centrifuge tube, then ti...

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Abstract

The invention discloses a multiplex-PCR detection method for a single sperm. The method comprises the steps that A, sperms are pretreated; B, micromanipulation is performed to pick up a single sperm; C, the sperm is subjected to multiplex-PCR detection. The multiplex-PCR detection method for a single sperm breaks through the limitation, only targeting female eggs and embryos, of past genetic disease gene detection on micro-cells and makes it possible for analysis of whether a male germ cell is a chimera. Through the method, very rare spontaneous mutation which only exists in a single sperm and cannot be detected in a somatic cell or sperm groups can be detected, auxiliary diagnosis analysis of de novo mutation can be performed on a paternal age effect series genetic disease caused by de novo mutation of a paternal germ cell, and on this basis, the risk of birth of a child patient with the genetic disease can be prevented and interdicted in combination with the preimplantation genetic diagnosis technology.

Description

technical field [0001] The invention relates to the field of PCR detection, in particular to a single sperm multiplex PCR detection method. Background technique [0002] At present, the detection of reproduction is mainly focused on women's pre-pregnancy, prenatal and newborns. Sperm is the only haploid that exists in men, and the detection of male germ cells is still in the exploratory stage. Only PCR detection of a single sperm is the best way to solve the inheritance of spontaneous mutations in germ cells. [0003] Because the sperm is haploid, the amount of DNA is only half of the somatic cells, only 3pg, and the chromatin density of the sperm head is very high, and the nuclear protein is mainly sperm nucleoprotein, which is very small, less than 1 / 20 of the egg . Therefore, stable single sperm PCR has always been a difficult problem. Contents of the invention [0004] Based on this, it is necessary to provide a single sperm multiplex PCR detection method for the ab...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q1/686C12Q2600/156C12Q2537/143C12Q2531/113
Inventor 刘刚李维娜卢光琇
Owner REPRODUCTIVE & GENETIC HOSPITAL OF CITIC XIANGYA CO LTD
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