Standard product for polyploid chromosome detection and preparation method thereof

A technology for standards and chromosomes, which is applied in biochemical equipment and methods, and the determination/inspection of microorganisms.

Active Publication Date: 2016-08-03
海南华大基因科技有限公司
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0006] Due to the incidence of trisomy 21 fetuses, trisomy 18 fetuses and trisomy 13 in the world is 1 / 600-1 / 800, 1 / 6000, 1 / 10000, based on 135,000,000 newborns per year worldwide In other words, considering the above-mentioned incidence rates of T21, T18, and T13, only 190,000, 22,000, and 13,000 positive pregnant women can be found in the world each year. Separation of plasma samples from pregnant women with trisomy 13 to construct standard products will face sampling difficulties, ethical disputes, limited blood collection, and large differences in fetal concentration among pregnant women, making it difficult to obtain standard products in the expected concentration range, etc. Therefore, the method of collecting real positive pregnant women's plasma samples to construct standards is poor in implementation

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  • Standard product for polyploid chromosome detection and preparation method thereof
  • Standard product for polyploid chromosome detection and preparation method thereof
  • Standard product for polyploid chromosome detection and preparation method thereof

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preparation example Construction

[0082] Preparation method of standard product

[0083] From a technical point of view, the present invention usually has the following four methods to prepare simulated samples:

[0084] 1) Genomic DNA extracted from known trisomy 21 patients or trisomy 21 cell lines is mixed with normal female genomic DNA in proportion, interrupted to the size distribution range of plasma DNA fragments, and set aside;

[0085] 2) Genomic DNA extracted from known trisomy 21 patients or trisomy 21 cell lines is used to break into the size distribution range of plasma DNA fragments, and mixed with the disrupted products of normal female genomic DNA in proportion;

[0086] 3) Genomic DNA extracted from patients with trisomy 21, 18, and 13 or cell lines with trisomy 21, 18, and 13 was used to break into the size distribution range of plasma DNA fragments, and plasma DNA separated from normal female peripheral blood mix in proportion;

[0087] 4) Plasma DNA isolated from peripheral blood of patie...

Embodiment 1

[0177] The influence of embodiment 1 different size trisomy DNA fragments on standard

[0178] This embodiment simulates the influence of three T21 gDNA fragment sizes (100-150bp, 150-200bp, 200-250bp) on the prepared plasma sample with abnormal fetal chromosome number when the fetal concentration is 10%.

[0179] The results are shown in Table 1. It can be seen from the results that the abnormal number of fetal chromosomes can be detected in the plasma samples prepared by DNA with three fragment sizes. Compared with the data, when the fragment size is selected as 150-200bp, the Z values ​​of the three platforms can all be tested. Stable above 3.50 and 150-200bp is the real situation of the fragment distribution size of free DNA in plasma. Based on the above considerations, it is preferred to choose trisomy gDNA with a fragment size of 150-200bp to interrupt and purify the DNA and mix it with normal female plasma to prepare the standard Taste.

[0180] Table 1

[0181]

...

Embodiment 2

[0183] The impact of embodiment 2 different polyploidy chromosome concentration on standard substance detection accuracy

[0184] According to the existing reports that triploids account for the percentage (3-20%) of the concentration of free DNA in the mother's plasma, using the general method, this embodiment simulates the concentration of the fetus as 1%, 2%, 3%, 3.5%, 4%, 5%. %, 7%, 10%, 12% and other 9 concentration gradients of T21 plasma samples to select the most suitable gradient as a standard.

[0185] The results can be seen in Table 2. Combined with the results of sequencing and information analysis, it can be seen that at about 3.5%, the Z value of chromosome 21 has a critical value, so about 3.5% can be selected as the minimum detection concentration value; and when the concentration is above 4%. , the Z value can show a relatively stable detection rate; when the fetal concentration is 10%, the detection rates of the three platforms are all 100%, and the simulate...

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Abstract

The invention provides a standard product for polyploid chromosome detection and a preparation method thereof. The standard product comprises a first standard, a second standard and an optional third standard which are respectively positioned inside a separate container, wherein the first standard contains 3.5+/-0.2% of polyploid chromosome gDNA fragments and residual normal female plasma on the basis of total mass (mass) of DNA in the standard; the second standard contains 4-6% of polyploid chromosome gDNA fragments and residual normal female plasma on the basis of total mass (mass) of DNA in the standard; and the first standard contains 8-12% of polyploid chromosome gDNA fragments and normal female plasma on the basis of total mass (mass) of DNA in the standard.

Description

technical field [0001] The present invention relates to second-generation high-throughput sequencing, and relates to the field of genetic variation detection, especially the detection of abnormal chromosome numbers, such as trisomy 21, trisomy 18, and trisomy 13. Background technique [0002] Trisomy 21 (also known as Down syndrome, T21), trisomy 18 (also known as Edwards syndrome, T18) and trisomy 13 (also known as Patau syndrome, T13), are Currently common birth defect chromosomal diseases, the newborn incidence rates are 1 / 600-1 / 800, 1 / 6000, 1 / 10000 respectively. At present, prenatal testing is mainly based on serum biochemical screening, with a false positive rate of about 3.2-5.6%, and a detection rate of only 66-81%. [0003] In 2008, it was reported that high-throughput sequencing technology was applied to the deep sequencing of the whole genome of free DNA in the plasma of pregnant women to detect whether the fetus suffers from chromosomal aneuploidy diseases, such ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 张艳艳陈芳蒋慧王逸丛蒋浩君徐讯
Owner 海南华大基因科技有限公司
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