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Trisome syndrome gene nucleic acid detection quality control product based on CRSIPR-Cas9 and organoid culture and preparation method therefor

A technology for trisomy syndrome and organoids, which is applied in the field of trisomy gene nucleic acid detection quality control products and its preparation, can solve the problems of large-scale clinical application and uneven precision, and achieve high genome stability, The effect of long storage time and high stability

Inactive Publication Date: 2021-02-23
苏州艾可瑞斯生物科技有限公司
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Problems solved by technology

However, such products have the characteristics of uneven precision between batches, which seriously hinders large-scale clinical application

Method used

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  • Trisome syndrome gene nucleic acid detection quality control product based on CRSIPR-Cas9 and organoid culture and preparation method therefor
  • Trisome syndrome gene nucleic acid detection quality control product based on CRSIPR-Cas9 and organoid culture and preparation method therefor
  • Trisome syndrome gene nucleic acid detection quality control product based on CRSIPR-Cas9 and organoid culture and preparation method therefor

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preparation example Construction

[0037] The invention provides a method for preparing a trisomy gene nucleic acid detection quality control product based on CRSIPR-Cas9 and organoid culture, and the preparation steps are as follows:

[0038] (1) Using CRISPR-Cas9 technology to knock-in and express telomerase TERT in peripheral blood lymphocytes derived from patients with trisomy 13, 18, and 21, to construct gene-edited lymphocytes that can stably expand and divide; the specific steps are as follows: :

[0039] Design 3 pairs of specific primers based on the full-length domain of the TERT gene, and amplify the relevant fragments to cover the entire CDS region of the TERT gene. The primers are shown in the table below (Table 1). After the amplification was completed, the three parts of the sequence amplified by the primers in Table 1 were integrated into a single Puro expression plasmid, and the expression donor plasmid with 800bp homology arms was inserted into the two segments respectively. Introduce CRISPR-...

Embodiment 1

[0056] Accuracy identification of quality control products for trisomy 13, 18, and 21 by next-generation sequencing

[0057] Use digital PCR technology to test different proportions of quality control products to identify their accuracy. The result is as Figure 5 As shown, in each order of magnitude, the digital PCR test results have a significant correlation with the expected mixing ratio, and the correlation coefficient is >0.95, confirming that the quality control product has a high degree of accuracy.

Embodiment 2

[0059] Reproducibility identification of quality control products for trisomy 13, 18, and 21 by next-generation sequencing

[0060] Full levels (1:10) prepared from three different batches using digital PCR technology 3 , 1:10 4 , 1:10 5 , 1:10 6 , 1:10 7 ) Quality control products are tested to identify the repeatability of different batches of products. The result is as Figure 6 As shown, there is no significant difference in the detection values ​​of the three different batches at all levels, and they are kept in a good SD range, which proves that the preparation process of this product has excellent repeatability.

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Abstract

The invention provides a trisome syndrome gene nucleic acid detection quality control product based on CRSIPR-Cas9 and organoid culture. The quality control product is applied to omni-directional monitoring on a trisome syndrome gene detecting process. A preparation method comprises the following steps: (1) carrying out telomerase TERT knock-in expression on peripheral blood lymphocytes derived from a trisome syndrome sufferer by using a CRISPR-Cas9 technology, so as to construct gene-edited lymphocytes capable of stably amplifying and dividing; (2) constructing a brand-new organoid culture system, carrying out large-scale amplification on the gene-edited lymphocytes, and verifying maintaining conditions of chromosomal aberration states of amplified cells through chromosomal karyotype analysis; and (3) mixing the product with wild type human-derived lymphocytes according to a specific proportion, and carrying out accurate quantitative verification, thereby preparing the trisome syndrome gene nucleic acid detection quality control product. A foundation biological genetic background is closer to that of an actual sufferer, the stability is higher compared with that of a genome of a quality control derived from an ordinary sufferer, and omni-directional monitoring on trisome syndrome gene detection is achieved.

Description

technical field [0001] The invention belongs to the field of nucleic acid detection of characteristic genes of trisomy 13, 18, and 21, and in particular relates to a trisomy gene nucleic acid detection quality control product based on CRSIPR-Cas9 and organoid culture and a preparation method thereof. Background technique [0002] Chromosomal trisomy syndrome is the enemy of current prenatal and postnatal care policies. The most common trisomy syndromes are 13, 18, or 21 trisomy syndromes. congenital disease. Most affected children will stop developing in early pregnancy, leading to miscarriage; however, a small number of fertilized eggs can still develop and mature, eventually leading to the birth of affected children. The intellectual development of the affected children was limited after birth, which was significantly lower than that of ordinary children of the same age. Due to the mental retardation and developmental delay of such children, it has caused a great burden ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6851C12N15/90C12N15/54C12N5/10C12N5/078
CPCC12Q1/6883C12Q1/6851C12N15/907C12N9/1276C12Y207/07049C12N5/0634C12Q2600/156C12Q2600/166C12N2510/04C12Q2531/113
Inventor 袁嘉扬吴炯马晓路
Owner 苏州艾可瑞斯生物科技有限公司
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