Separation and purification method for fetal nucleated red blood cells and Down syndrome screening kit

A technology for Down's syndrome and nucleated red blood cells, applied in the field of separation and purification of fetal nucleated red blood cells and Down's syndrome screening kits, can solve the problem of fetal cell enrichment, small number of separation, insufficient purity, and no fundamental breakthrough And other issues

Inactive Publication Date: 2013-05-15
SHANDONG YADA PHARMA
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Problems solved by technology

[0007] To sum up, the current domestic and foreign researches on this aspect all have the problems of enrichment of fetal cells, small number of separation, insufficient purity, complicated operation process, and very limited range of diseases applied to prenatal diagnosis, making this work fundamentally impossible. breakthrough

Method used

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  • Separation and purification method for fetal nucleated red blood cells and Down syndrome screening kit
  • Separation and purification method for fetal nucleated red blood cells and Down syndrome screening kit
  • Separation and purification method for fetal nucleated red blood cells and Down syndrome screening kit

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Embodiment 1

[0018] 1. Isolation and purification of fetal NRBC and DNA extraction

[0019] Take 2-5ml of peripheral venous blood from pregnant women at 10-18 weeks of pregnancy, add it to a heparin anticoagulation tube containing 10E+8 anti-CD45 antibody magnetic beads for negative screening, mix well to absorb white blood cells, and then put it on a magnetic separator Separation for 5 minutes, transfer the upper layer of blood to a heparin anticoagulant tube containing 10E+7 anti-CD71 antibody magnetic beads for positive screening, and fully wash with phosphate buffered saline (PBS) to obtain fetal NRBC; then separate on a magnetic separator for 5 minutes After removing the clean PBS, add ultrapure water, lyse the cells at 98°C for 5 minutes, release the DNA, and store in a -20°C refrigerator for later use.

[0020] 2. Use the specific sequence of chromosome 21 DSCR segment sequence and the homologous sequence USC2 on chromosome 2 to synthesize its shared primers and fragment-specific tw...

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Abstract

The invention discloses a separation and purification method for fetal nucleated red blood cells and a Down syndrome screening kit and belongs to the detection field of molecular cell biology. The separation and purification method comprises the following steps: firstly, fully mixing maternal peripheral blood and a CD45 antibody labeling magnetic bead to adsorb white blood cells; discarding on a magnetic separator, transferring upper-layer cells to a test tube containing a CD71 antibody labeling magnetic bead, and fully and uniformly mixing; separating on the magnetic separator to obtain nucleated red blood cells; extracting a genome DNA (Deoxyribonucleic Acid) of the nucleated red blood cells; adding SRY (Sexdetermining Region Y), DSCR (Down Syndrome Critical Region) and USC2 specific probes, DSCR and USC2 competitive primers, an SRY specific primer and a DNA to be detected into the same competitive fluorescent quantitation PCR (Polymerase Chain Reaction) reaction system for multiple competitive real-time fluorescence quantification PCR reactions to obtain Cts of DSCR and USC2; and calculating a difference value between the Cts to judge whether a sample to be detected is highly risk to DS (Down Syndrome).

Description

technical field [0001] The invention relates to a kind of separation and purification of nucleated red blood cells (NRBC) and the preparation of a rapid gene screening kit, belonging to the field of molecular cell biology detection, in particular to a kind of separation and purification of fetal NRBC in the peripheral blood of pregnant women and the use of NRBC for Down's syndrome Syndrome (Down's syndrome, DS) rapid genetic detection kit can be applied to prenatal screening of DS. Background technique [0002] Down syndrome, also known as congenital stupidity, trisomy 21. The incidence of fetal DS in pregnant women is 1 / 100, and most of them are aborted in the early stage. In newborns, its incidence rate is 1 / 600-800, and it increases with the age of pregnant women. Every pregnant woman has a certain risk. It is the most common chromosomal abnormality disease in humans. Its clinical manifestations are: Mental retardation (IQ between 25-30%), congenital multiple deformitie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078C12Q1/68
Inventor 夏庆杰杨林吴苹
Owner SHANDONG YADA PHARMA
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