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Reference product for non-invasive prenatal detection of fetal aneuploid chromosomes

A technology for aneuploidy and prenatal detection, applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc. Easy quality control, important application prospects, easy to prepare results

Active Publication Date: 2019-01-01
国家卫生健康委科学技术研究所 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the development of this technology is currently limited by the acquisition of reference products. Currently, there is no reference product for non-invasive prenatal testing technology, and no available reference products are available in the market, which seriously restricts the development of related products and the quality control of existing products. Therefore, the present invention provides a reference product for non-invasive prenatal detection of fetal aneuploid chromosomes, which is used for the research and development and quality control of non-invasive prenatal detection related products

Method used

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  • Reference product for non-invasive prenatal detection of fetal aneuploid chromosomes
  • Reference product for non-invasive prenatal detection of fetal aneuploid chromosomes
  • Reference product for non-invasive prenatal detection of fetal aneuploid chromosomes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1 prepares positive reference substance by the genomic DNA of tissue and blood source

[0023] Sample selection: Sample 1 is a placental tissue sample of a T21 trisomy (Down syndrome) positive male fetus. The reason for selecting a male fetus is to facilitate the quantitative detection of the incorporation ratio of mixed nucleic acids; Sample 2 is a maternal sample, that is, the fetus Maternal peripheral blood, two samples genetically related, was used to simulate the composition of cell-free DNA derived from maternal peripheral blood. Sample 3 is a plasma sample of a pregnant woman, which is used to provide plasma cell-free DNA.

[0024] DNA extraction: Genomic DNA of sample 1 (placental tissue sample) was extracted using a genome extraction kit (DP304, Tiangen Biochemical Technology Beijing Co., Ltd.) according to the instructions. For sample 2 (peripheral blood), red blood cells were first lysed, and then the genomic DNA was extracted from the isolated wh...

Embodiment 2

[0031] Example 2 Prepare positive reference substance by culturing the genomic DNA derived from cells

[0032] Sample selection: sample 4 is the T21 trisomy-positive cell line AG09394 (male), and sample 5 is the corresponding mother's cell line AG09387. Use RPMI 1640 medium, containing 2mM L-glutamine, 15% fetal bovine serum for cell culture, add 10-20ml medium (vertical placement culture) to T25 tissue culture flask, 37 ° C, CO2 concentration 5% incubator culture . Keep the cell concentration in the medium not lower than 200,000 cells / ml. Fresh medium should be replaced every 3-4 days. The upper limit of culture density is 1 million cells / ml, count the cells after culture, and then count 1×10 8 Cell Genome Extraction Kit (DP304, Tiangen Biochemical Technology Beijing Co., Ltd.) was used to extract the cellular genomic DNA of samples 4 and 5 according to the instructions.

[0033] DNA Fragmentation: Fragmentation of DNA and purification of magnetic beads according to the m...

Embodiment 3

[0037] Example 3 Detection of T21 Positive Reference Substances by Noninvasive Prenatal DNA Detection Reagents

[0038] In order to verify whether the prepared positive reference product for non-invasive prenatal detection of fetal aneuploid chromosomes can meet the needs of non-invasive prenatal detection, this experiment uses the non-invasive prenatal next-generation sequencing kit (Boao Bio) to test it. The test samples are plasma free DNA and two fragmented DNA mixtures in Example 1 and Example 2.

[0039] First, the library was constructed according to the instructions, and then the library concentration was tested by qPCR. The library concentration results are shown in Table 2. The data in Table 2 shows that there is no difference in the library concentration among the three samples, and the library construction results meet the test expectations.

[0040] Then the library was sequenced and analyzed, and the above three samples were sequenced according to the normal SOP ...

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Abstract

The present invention provides a reference product for non-invasive prenatal detection of fetal aneuploid chromosomes. The reference product comprises: a first fragmented nucleic acid derived from thetissue of a maternal body having normal chromosome ploidy or the cultured cell line genome, and a second fragmented nucleic acid derived from the tissue or the cultured cell line genome of a fetus with positive chromosome aneuploidy, wherein the first fragmented nucleic acid is genetically related to the second fragmented nucleic acid, the first fragmented nucleic acid at least accounts for 70% of the total nucleic acid amount of the reference product, and the second fragmented nucleic acid accounts for 5-30% of the total nucleic acid amount of the reference product. According to the presentinvention, the reference product is easy to prepare, meets clinical practical demands, is used for the research and development and quality control on non-invasive prenatal detection related products,and has important application prospects.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a reference product for non-invasive prenatal detection of fetal aneuploid chromosomes. Background technique [0002] Improving the level of reproductive health and preventing and controlling major birth defects are major needs in my country. With the liberalization of the two-child policy, there will be more elderly pregnant women. How to carry out prenatal testing accurately, simply, quickly and effectively to control birth defects is always on the table of relevant government departments and clinical testing laboratories in our country. A difficult problem ahead. Early prenatal screening and diagnosis is the most effective response to birth defects. At present, traditional prenatal diagnosis techniques such as amniocentesis, chorionic villus biopsy, and cord blood extraction have inherent defects that cannot be overcome by the invasive sampling method itself, and there are cons...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/166
Inventor 祝令香高华方陈西华曹宗富郭永陆超刘烨杨文军王勇斗
Owner 国家卫生健康委科学技术研究所
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