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Circulating tumor dissociating DNA mutation detection quality control products and preparation method thereof

A technology for mutation detection and quality control, applied in the fields of clinical laboratory science and biology and medicine

Inactive Publication Date: 2018-09-11
ANNOROAD GENE TECH BEIJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no such quality control product in China to evaluate each laboratory

Method used

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  • Circulating tumor dissociating DNA mutation detection quality control products and preparation method thereof
  • Circulating tumor dissociating DNA mutation detection quality control products and preparation method thereof
  • Circulating tumor dissociating DNA mutation detection quality control products and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Preparation of quality control products for ctDNA mutation detection

[0045] 1. Method

[0046] 1. Cultivate human normal cell-derived cell lines and extract genomic DNA

[0047] (1) Cell culture: human normal cell-derived cell line HEK293T was cultivated in DMEM medium containing 10% fetal bovine serum, which contained 10% fetal bovine serum, 100IU / ml penicillin and 100IU / ml streptomycin; at 37 Cultivate in a constant temperature cell incubator containing 5% CO2. Cell subculture was digested with trypsin, and the cells were expanded to 10 7 ~10 8 and in the logarithmic growth phase.

[0048] (2) Extraction of human genomic DNA: According to the instructions of the kit (QIAmp DNA mini kit, QIAGENE, China), the genomic DNA product of the human normal cell-derived cell line HEK293T was obtained and stored at -20°C.

[0049] 2. Cultivate cell lines derived from human normal cells, and digest the cells with MNase to obtain fragmented genomic DNA

[0050] (...

Embodiment 2

[0081] Example 2: Validation of quality control products for ctDNA mutation detection using the dPCR method

[0082] 1. Method

[0083] Validation using the Bio-Rad QX200Droplet Digital PCR System

[0084] Take a set of quality control sample trays obtained in Example 1, and treat it as a conventional ctDNA sample for dPCR testing at Shanghai Austai Medical Laboratory, and obtain the test results through steps such as system preparation, on-board testing, and data analysis.

[0085] In this implementation case, the dPCR detection range is the gene mutation site in the following table:

[0086] Table 7

[0087]

[0088]

[0089] 2. Results

[0090] See Table 8.

[0091] Table 8

[0092]

[0093] The test results of this implementation case show that the Bio-Rad QX200DropletDigital PCR system of Shanghai Austai Medical Laboratory can detect most of the mutations in the quality control sample plate, and the reporting frequency is basically in line with the expectati...

Embodiment 3

[0094] Example 3: Validation of quality control products for ctDNA mutation detection using the ARMS method

[0095] 1. Method

[0096] The Mx 3000P fluorescent PCR instrument of Agilent Company was used for verification.

[0097] Take a set of quality control sample trays obtained in Example 1, and treat it as a conventional ctDNA sample for ARMS testing at Xiamen Aide Medical Laboratory, and obtain the test results through system preparation, on-machine testing, and data analysis.

[0098] In this implementation case, the detection range of ARMS is the gene mutation site in the following table:

[0099] S table 9

[0100]

[0101]

[0102] 2. Results

[0103] See Table 10.

[0104] Table 10

[0105]

[0106] The test results of this implementation case show that Xiamen Aide Medical Laboratory can detect most of the mutations in the quality control sample plate by using the Mx 3000P fluorescent PCR instrument, that is, the ctDNA mutation detection quality contro...

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Abstract

The invention discloses circulating tumor dissociating DNA mutation detection quality control products and a preparation method thereof, and belongs to the technical field of medical science, clinicallaboratory science and biology. The ctDNA mutation detection quality control product consists of genomic DNA after enzyme digestion and ultrasound-interrupted DNA fragments containing tumor-associated gene mutation. A method used for preparing EQA quality control products is provided by the ctDNA mutation detection quality control product obtained by mixed production of the ultrasound-interruptedDNA fragments containing different gene mutation and the genomic DNA after the enzyme digestion. The preparation method is still the first case.

Description

technical field [0001] The invention relates to a quality control product for detection of circulating tumor free DNA and a preparation method thereof, in particular to a quality control product prepared based on ultrasonic interruption and enzymatic digestion methods, which can be used for mutation detection of various circulating tumor free DNA and its preparation The method belongs to the fields of medicine, clinical laboratory science and biotechnology. Background technique [0002] Circulating tumor DNA (ctDNA) is a single-stranded or double-stranded DNA molecule that often exists in plasma or serum. It was first discovered in the peripheral blood of tumor patients in 1977. In the pre-genome era, the characteristics of ctDNA could not be studied due to technical limitations. In the ensuing 17 years, people gradually understood some of the biological characteristics of ctDNA, such as its possible release mechanism, possible degradation mechanism, fragment size distribu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6806C12N15/11C12N15/10
CPCC12Q1/6806C12Q1/6886C12Q2600/156C12Q2600/166C12Q2521/301C12Q2531/113C12Q2523/301
Inventor 李金明彭绒雪张瑞
Owner ANNOROAD GENE TECH BEIJING
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