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FFPE reference substance as well as preparation method and application thereof

A reference material and cell technology, applied in the field of its preparation and FFPE reference material, can solve the problem of not being able to play an accurate reference role, and achieve the effect of improving accuracy

Inactive Publication Date: 2020-08-11
北京橡鑫生物科技有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The main purpose of the present invention is to provide a FFPE reference product, its preparation method and application, to solve the problem that the FFPE reference product in the prior art cannot play an accurate reference role

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  • FFPE reference substance as well as preparation method and application thereof
  • FFPE reference substance as well as preparation method and application thereof
  • FFPE reference substance as well as preparation method and application thereof

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preparation example Construction

[0024] In a typical embodiment of the present application, a method for preparing FFPE reference products is provided. The preparation method includes: selecting a target cell line; after culturing the cells of the target cell line to a predetermined concentration, collecting the cells for formal Lin fixed and made paraffin blocks; sliced ​​the paraffin blocks to obtain FFPE reference products.

[0025] The preparation method of the FFPE reference product is to select a suitable cell line, then culture and prepare the same FFPE sample as the clinical sample, so as to provide the clinical FFPE sample with a reference comparison function from nucleic acid extraction to test results. It is an FFPE sample The entire testing process plays a quality control role, thereby improving the accuracy of the testing results. It is suitable for mass preparation of FFPE reference materials for tumor cell lines containing specific gene mutation ratios, which solves the problem that enterprises do...

Embodiment 1

[0043] For the EGFR gene, prepare FFPE positive reference samples for 4 mutation sites of EGFR gene p.G719S, Exon19Del, p.S768I, and p.L858R, including the following steps:

[0044] (1) Purchasing cell lines, the specific information is as follows:

[0045] Table 1:

[0046]

[0047] (2) Cell culture, DNA extraction of cultured cell lines.

[0048] (3) Use QuantStudio for the extracted cell line TM 3D Digital PCR performs verification to determine the mutation frequency of the site.

[0049] As shown in the following table:

[0050] Table 2:

[0051] Serial number Cell line name Mutation name Digital PCR detection frequency Digital PCR scatter plot 1 SW48 p.G719S27.43％ figure 1 2H1650 E746_A750del60.07％ figure 2 3 KYSE450 p.S768I49.70％ image 3 4H1975 p.L858R67.85％ Figure 4

[0052] (4) The cells were diluted by the limiting dilution method, and the monoclonal cells containing the target gene mutation were selected for culture, and counted at 12h, 24h, 36h, 48h and 60h respectively, a...

Embodiment 2

[0064] Preparation of wild-type FFPE cell line reference:

[0065] (1) Purchasing cell lines, the specific information is as follows:

[0066] table 5:

[0067]

[0068]

[0069] (2) Cell culture, DNA extraction of the cultured cell line, Sanger sequencing of the extracted genomic DNA to verify the gene mutation.

[0070] (3) After the verification is passed, the wild-type cell line will be enlarged and cultivated, and the number of cells will reach 4×10 6 ~5×10 6 When cells / mL, digest the adherent cells and collect the cells by centrifugation.

[0071] (4) Cell embedding and paraffin roll preparation

[0072] The collected cells were washed 3 times with PBS and then fixed with 10% formalin for 2 hours, and processed according to the conventional dehydration procedure. After the dehydration procedure is completed, the tissue block is taken out from the automatic dehydrator and embedding in conventional paraffin. Put the embedded cell line wax blocks on the freezing table and freeze the...

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Abstract

The invention provides an FFPE reference substance as well as a preparation method and application thereof. The preparation method of the FFPE reference substance comprises the following steps: selecting a target cell line; culturing the cells of the target cell line to a predetermined concentration, collecting the cells, and carrying out formalin fixation to prepare a paraffin block; and slicingthe paraffin block to obtain the FFPE reference product. According to the method, the FFPE sample which is the same as a clinical sample is prepared by selecting a proper cell line and culturing, so that a reference comparison effect from nucleic acid extraction to a detection result can be provided for the clinical FFPE sample, a quality control effect is achieved for the whole process of FFPE sample detection, and the accuracy of the detection result is improved. The method is suitable for large-batch preparation of tumor cell strain FFPE reference substances containing specific gene mutation proportions, so that the problem that no full-process FFPE reference substances exist in internal quality control during research, development and clinical examination of products such as kits prepared by enterprises is solved.

Description

Technical field [0001] The present invention relates to the field of FFPE sample detection, in particular to a FFPE reference product, its preparation method and application. Background technique [0002] In recent years, with the continuous development of molecular biology technology, especially the wide application of PCR technology and high-throughput sequencing technology, genetic testing technology has gradually moved from laboratory to clinical application, especially in cancer treatment. They are all related to specific gene mutation types. Therefore, in the development and analytical performance verification of genetic testing products, enterprise reference products containing different mutation frequencies of specific genes are required to evaluate the sensitivity, accuracy, specificity and Repeatability. At the same time, in the process of genetic testing by third-party clinical laboratories, reference materials for specific mutation frequencies of certain genes are al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N1/36C12N5/09
CPCC12N5/0693G01N1/286G01N1/30G01N1/36G01N2001/2873
Inventor 楼峰孙宏曹善柏
Owner 北京橡鑫生物科技有限公司
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