FFPE reference product for gene detection and preparation method and application of FFPE reference product

A technology for genetic testing and reference materials, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., which can solve the problems of small number of clinical FFPE samples, irregular frequency and site

Pending Publication Date: 2019-04-16
ZHENYUE BIOTECHNOLOGY JIANGSU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The present invention aims to provide a FFPE reference product for gene detection, its preparation method and application, so as to solve the te...

Method used

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  • FFPE reference product for gene detection and preparation method and application of FFPE reference product
  • FFPE reference product for gene detection and preparation method and application of FFPE reference product
  • FFPE reference product for gene detection and preparation method and application of FFPE reference product

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preparation example Construction

[0020] According to a typical embodiment of the present invention, a method for preparing an FFPE reference product for gene detection is provided. The preparation method comprises the following steps: S1, performing cell culture on tumor cell lines, and collecting cell pellets; S2, performing formalin fixation on cell pellets, and then wrapping the cell pellets with agarose gel to form cell clusters, and making the cell clusters into a cell wax block; S3, extract the genomic DNA of the tumor cell line in the cell wax block, and measure the gene mutation frequency of the genomic DNA of the tumor cell line; S4, mix the genomic DNA of the tumor cell line containing the target mutation site into a The DNA mixture of the target mutation frequency, the DNA mixture is the FFPE reference product for genetic detection.

[0021] Applying the technical scheme of the present invention, formalin-fixed paraffin embedding is carried out on the cells cultured by tumor cell lines, which can b...

Embodiment approach

[0033] According to a typical embodiment of the present invention, there are one or more target mutation sites.

[0034] In a typical embodiment of the present invention, the tumor cell line is centrifuged and the supernatant is discarded to collect the cells. After washing with PBS for 2-3 times, fix the cells with 10% formalin for 1 hour, collect the cells by centrifugation, discard the formalin, wash the cells with PBS for 2-3 times, then wrap the cells with agarose gel to form a pellet Cell clumps. The agarose gel preferably has a concentration of 2%, a temperature of 60° C., and a volume of 30 μL.

[0035] In a typical embodiment of the present invention, the production of cell wax block is preferably fixed with 10% neutral formaldehyde for 2 hours, and processed according to a conventional dehydration procedure. After the dehydration procedure is completed, the tissue block is taken out from the automatic dehydrator, and is preferably subjected to conventional paraffin...

Embodiment 1

[0043] The method for preparing lung cancer and colorectal cancer hotspot mutation formalin-fixed paraffin-embedded reference product comprises the following steps:

[0044] 1) Cultivate the cell lines purchased from Nanjing Kebai. The 21 cell lines involved are as follows: AMO-1, NCI-H929, A549, A-427, HCT116, THP-1, NCI-H2347, SK-HEP -1, PC-9, NCI-H1975, KYSE450, SW48, NCI-H2228, NCI-H3122, HCC78, NCI-N87, EBC-1, NCI-H1299, NCI-H460, SW948, GM12878. The specific information about the cell lines is shown in Table 1:

[0045] Table 1

[0046]

[0047] 2) Genomic DNA was extracted from the cultured cell line, using the QIAamp DNA Mini Kit (Product No.: 51304) nucleic acid extraction kit produced by Kaijie Enterprise Management (Shanghai) Co., Ltd.

[0048] 3) The DNA of the extracted tumor cell lines was verified by first-generation sequencing, so as to clarify the mutation background of the cell lines.

[0049] 4) Prepare the cell line wax block, the specific method is a...

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Abstract

The invention discloses an FFPE reference product for gene detection and a preparation method and application of the FFPE reference product. The preparation method comprises the following steps that S1, cell culture is performed on a tumor cell line, and cell pellets are collected; S2, formalin fixation is performed on the cell pellets, then sepharose gel wraps the cell pellets to form cell aggregates, and the cell aggregate are prepared into cell paraffin blocks; S3, genomic DNA of the tumor cell line in the cell paraffin blocks is extracted, and genetic mutation frequency determination is performed on the genomic DNA of the tumor cell line; S4, the genomic DNA of the tumor cell line containing a target mutation site is mixed into a DNA mixture of a target mutation frequency, and the DNAmixture is the FFPE reference product for gene detection. According to the technical scheme, formalin fixation and paraffin wrapping are performed on the cells cultured by tumor cell line, so that thesituation of clinical samples can be better simulated.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to an FFPE reference product for gene detection, its preparation method and application. Background technique [0002] In recent years, the field of genetic testing has developed extremely rapidly, involving early screening of cancer, diagnosis, adjuvant therapy, and detection and analysis of individualized treatment of other diseases. At present, the main detection methods used are next-generation sequencing, ARMS PCR, ddPCR technology, etc. Correspondingly, many such genetic detection kits have appeared on the market. [0003] Clinical-based genetic testing kits need to use reference materials in the research and development process. Reference materials are important methods and indicators for verifying and evaluating the accuracy and stability of kit detection. [0004] Most of the clinical samples are formalin-fixed paraffin-embedded samples (FFPE samples, Formalin-Fixed...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156C12Q2600/166
Inventor 王晓红林小静洪媛媛闫慧婷王海波陈维之
Owner ZHENYUE BIOTECHNOLOGY JIANGSU CO LTD
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