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Method of prenatal gene screen for down's syndrome using nucleated erythrocyte and kit

A technology for Down syndrome and nucleated red blood cells, which is applied in the field of fluorescent quantitative PCR kits, can solve the problems of fetal cell enrichment, small number of isolated cells, no fundamental breakthrough, limited disease scope, etc., and achieves simple and fast operation. , The effect of low labor cost and high result accuracy

Inactive Publication Date: 2009-02-04
SHANDONG YADA PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 separations, low purity, complicated operation process, and the scope of diseases applied to prenatal diagnosis is very limited, which makes this work impossible. fundamental breakthrough

Method used

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  • Method of prenatal gene screen for down's syndrome using nucleated erythrocyte and kit
  • Method of prenatal gene screen for down's syndrome using nucleated erythrocyte and kit
  • Method of prenatal gene screen for down's syndrome using nucleated erythrocyte and kit

Examples

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Embodiment 1

[0043] (1) Isolation and purification of fetal NRBC and DNA extraction

[0044] Take 2ml of peripheral venous blood from pregnant women at 10-14 weeks of pregnancy, anticoagulate with EDTA, gently add to the top of Percoll in a 10ml centrifuge tube containing 2ml of Percoll cell separation solution (Pharmaci), centrifuge at 300g for 30min, collect and extract 1.075g / The peripheral blood nucleated cell fraction in the Percoll density range from ml to 1.085g / ml was mixed evenly with 100ul (containing about 10E6 antibody magnetic beads) and PBS containing anti-CD71 antibody magnetic beads, and incubated at 4°C for 1 hr, and placed on a magnetic separator Let it stand for 5 minutes, discard the liquid, add 1ml of phosphate buffered saline (PBS) to wash fully for 2 minutes, put it on the magnetic separator and let it stand for 5 minutes, discard the liquid, then wash it fully with 500ul PBS for 2 minutes, put it on the magnetic separator and let it stand for 5 minutes, discard As ...

Embodiment 2

[0074] (1) Isolation and purification of fetal NRBC and DNA extraction

[0075] Take 5ml of peripheral venous blood from pregnant women at 14-18 weeks of pregnancy, anticoagulate with EDTA, gently add to the top of Percoll in a 10ml centrifuge tube containing 5ml of Percoll cell separation solution (Pharmaci), centrifuge at 300g for 30min, collect and extract 1.075g / The peripheral blood nucleated cell fraction in the Percoll density range from ml to 1.085g / ml was mixed evenly with 100ul (containing about 10E6 antibody magnetic beads) and PBS containing anti-CD71 antibody magnetic beads, and incubated at 4°C for 1 hr, and placed on a magnetic separator Let it stand for 5 minutes, discard the liquid, add 1ml of phosphate buffered saline (PBS) to wash fully for 2 minutes, put it on the magnetic separator and let it stand for 5 minutes, discard the liquid, then wash it fully with 500ul PBS for 2 minutes, put it on the magnetic separator and let it stand for 5 minutes, discard As ...

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Abstract

The invention relates to a prenatal gene screening method for Down's syndrome by using nucleated erythrocyte and a kit thereof. The method is that a kit which comprises a nucleated erythrocyte purification reagent, primers and a bichrome Taqman fluorescent probe which are synthesized by the specific sequence DSCR section sequence of chromosome 21 and the GAPDH gene sequence on chromosome 16, and an amplification buffering action reagent of PCR action. The invention realizes that through the following steps: (a) the purification of fetus NRBC and the extraction of DNA; (b) the synthesis of the primers and the bichrome Taqman fluorescent probe respectively by using the specific sequence DSCR section sequence of chromosome 21 and the GAPDH gene sequence on chromosome 16; (c) the co-amplification of two pairs of the primers to obtain the curve of fluorescence quantitative PCR by the bichrome fluorescence quantitative PCR reaction in the amplification buffering reaction system. The invention has simple and convenient operation and high testing throughput, and belongs to a non-invasive prenatal diagnostic method.

Description

technical field [0001] The invention relates to a method and kit for prenatal genetic screening of Down's syndrome by using nucleated red blood cells (NRBC), belonging to the field of molecular cell biology detection. Specifically, it relates to a fluorescent quantitative PCR kit for detecting the ploidy of chromosome 21 by separating and purifying fetal NRBC in the peripheral blood of pregnant women, which can be applied to prenatal screening of genetic material abnormalities such as Down's syndrome. technical background [0002] Down syndrome (Down Syndrome, DS), also known as congenital stupidity, is one of the most common chromosomal abnormal syndromes in live births, with an incidence rate of about 1 / 600-1 / 800. The signs of DS are very diverse, usually involving abnormalities of various tissues and organs, but its most prominent and severe clinical manifestation is mental retardation. The main clinical features of the disease are: severe mental retardation, and the phe...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 夏庆杰杨林
Owner SHANDONG YADA PHARMA
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