Gene group and kit for diagnosing lung caner, and diagnosis method thereof

A lung cancer diagnosis and kit technology, applied in the fields of genetic engineering and biotechnology detection, can solve the problems of less tumor-related gene capture, missing gene mutation information, low sequencing sensitivity and accuracy, and optimize the detection process and improve the detection accuracy. Effect

Inactive Publication Date: 2017-12-15
天津脉络医学检验有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The problem of the prior art that the present invention solves is: for existing lung cancer detection method usually has the following defect and deficiency: first, traditional detection method, comprises surgical operation treatment, radiotherapy and chemotherapy etc., can't detect tumor early Related symptoms; second, the existing detection methods based on lung cancer genes usually require a relatively high initial amount of library construction, and the sequencing sensitivity and acc...

Method used

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  • Gene group and kit for diagnosing lung caner, and diagnosis method thereof
  • Gene group and kit for diagnosing lung caner, and diagnosis method thereof
  • Gene group and kit for diagnosing lung caner, and diagnosis method thereof

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preparation example Construction

[0044] In a preferred embodiment of the present invention, the present invention provides the following preparation method, comprising:

[0045] (1) Preparation of DNA samples: Extract free DNA from plasma of different samples for library construction;

[0046] (2) End repair of the DNA sample: performing end repair on the free DNA obtained in step (1);

[0047] (3) Magnetic bead purification: use magnetic beads to purify the end-repaired DNA sample, preferably use AMPure XP magnetic beads for purification;

[0048] (4) Connect the A tail: connect the A tail to the purified product;

[0049] (5) Sample DNA connection adapter: connect the sample DNA product connected by the A tail to the connection adapter;

[0050] (6) performing library amplification on the sample DNA ligated by the adapter in step (5) to obtain a pre-capture PCR sample;

[0051] (7) Library hybridization: use specific probes to hybridize with PCR samples before capture to obtain ctDNA, and wash to obtain ...

Embodiment 1

[0061] according to figure 1 As shown in the flow chart, sample extraction, library construction, on-machine sequencing and data analysis were performed respectively. The specific process is as follows:

[0062] 1. Extraction of cell-free DNA

[0063] Using the ctDNA extraction kit (article number: 154022516, manufacturer: QIAGEN), the free DNA in the plasma of 4 different samples (these samples come from different tumor patients) can be extracted at the same time, and the DNA is quantified and dissolved in 50 μL ddH 2 In O, library construction was performed. The required reagent preparations are shown in Table 2.

[0064] Reagents required in Table 2

[0065] Reagent name

storage method

preparation method

Buffer ACL*

room temperature

room temperature

Buffer ACW1

room temperature

room temperature

Buffer ACW2

room temperature

room temperature

Buffer ACB

room temperature

room temperature

Pr...

Embodiment 2

[0198] Taking CBR3 and CDA genes as examples, the method of multiplex PCR and the method of probe capture sequencing described in Example 1 were respectively studied for CBR3 and CDA genes, and library construction and sequencing were performed on the amplified and captured samples. The sample used was the product of cell-free DNA extraction from lung cancer plasma, and the initial concentration of cfDNA was 1.4 ng / μL.

[0199] Different primer sequences used in different exon regions of different genes in table 16

[0200]

[0201]

[0202] Using the NimbleGEN SeqCap Hybridization and Wash Kit kit, multiplex PCR (30-35 circles) was used to amplify the target fragment, and the concentration of the product was detected and the library was sequenced. At the same time, the high-throughput sequencing method of Example 1 was used to construct the library and perform capture sequencing. see results Figure 5-Figure 8 .

[0203] in Figure 5 and Image 6 For the statistica...

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Abstract

The invention relates to the field of genetic engineering and biotechnologic detection, and concretely relates to a gene group and a kit for diagnosing lung cancer, and a diagnosis method thereof. The gene group for diagnosing the lung cancer includes ABCB1, AKT1, ALK, APC, ATIC and other 63 genes. The method using the gene group to diagnose the lung cancer comprises the following steps: (1) extracting free DNA and genome DNA from the plasma of a sample to be detected; and (2) breaking the genome DNA into fragments with the length of 150-250 bp, carrying out hybrid capture on the broken genome DNA and the free DNA to construct a DNA library, carrying out online sequencing, and analyzing the obtained sequencing result. Exon and partial intron regions of the 68 genes of the free DNA are enriched at one time by a probe capture technology to realize multi-gene and multi-target parallel deep high-throughput sequencing with high accuracy, optimize the detection flow and improve the detection precision, so liquid biopsy, low-frequency detection and tumor diagnosis become possible.

Description

technical field [0001] The invention relates to the fields of genetic engineering and biotechnology detection, in particular to a gene group, a kit and a diagnostic method for lung cancer diagnosis. Background technique [0002] Circulating DNA is a kind of extracellular DNA in a cell-free state, which exists in body fluids such as blood, synovial fluid, and cerebrospinal fluid. It is mainly composed of single-stranded or double-stranded DNA and a mixture of single-stranded and double-stranded DNA. Complex or free DNA exists in two forms. As a new tumor marker, circulating DNA will play an important role in the diagnosis, treatment and prognosis of tumors, especially for some tumors that do not have typical clinical symptoms, non-specific examination and difficult diagnosis can avoid complex, Invasive biopsy. [0003] Circulating tumor DNA (ctDNA) refers to the somatic DNA of tumor cells that is shed or released into the circulatory system after apoptosis. With the rapid d...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C40B50/06
CPCC12Q1/6869C12Q1/6886C12Q2600/156C40B50/06C12Q2535/122
Inventor 刘鹏飞王晓巍
Owner 天津脉络医学检验有限公司
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