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Method for extracting cfDNA (cell-free deoxyribonucleic acid) from hydrothorax, kit and constructed cfDNA library

A kit and pleural effusion technology, applied in the field of cfDNA library, can solve problems such as affecting detection, and achieve the effect of solving the problems of sample singleness and detection rate

Active Publication Date: 2017-06-20
NANJING SHIHE MEDICAL DEVICES CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the detection of mutations in plasma samples is affected by many factors. The state of the tumor, such as whether the patient is currently stable or progressing, will affect the detection of tumor-specific mutations. Such cancer stages, cancer metastasis sites, etc. may affect the plasma The detection of ctDNA in the middle, so there is an urgent need for a supplement of liquid biopsy samples

Method used

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  • Method for extracting cfDNA (cell-free deoxyribonucleic acid) from hydrothorax, kit and constructed cfDNA library
  • Method for extracting cfDNA (cell-free deoxyribonucleic acid) from hydrothorax, kit and constructed cfDNA library
  • Method for extracting cfDNA (cell-free deoxyribonucleic acid) from hydrothorax, kit and constructed cfDNA library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Obtaining cfDNA in pleural effusion samples

[0077] 1 Separation of supernatant and sediment from pleural fluid samples

[0078] Take the freshly collected pleural effusion sample, centrifuge at 1800rpm for 10 min, absorb the pleural effusion supernatant into a new centrifuge tube, discard the pleural effusion sediment, and store the pleural effusion supernatant sample in a refrigerator at 4°C (-80°C).

[0079] 2 Column adsorption separation of cfDNA in pleural effusion supernatant

[0080] 2.1 Sampling: Take out the pleural fluid supernatant sample stored at 4°C (-80°C), and then process it at 16000rpm at 4°C for 10 min;

[0081] 2.2 Prepare the premix: prepare the lysate Buffer ACL Mix (QIAGEN), draw 2 times the total volume of pleural effusion of BufferACL Mix, invert and mix well;

[0082] 2.3 Add proteinase K: add 20 μl Carrier RNA to each ml of pleural fluid sample, then add 200 μl proteinase K to fully digest;

[0083] 2.4 Add ACL: Add 2 ml of lysis...

Embodiment 2

[0105] The construction of embodiment 2 DNA library

[0106] 3.1 Separation of cfDNA size fragments

[0107] a) Add enzyme-free water to the cfDNA sample extracted in Example 1 to make up to 50 μl, add 35 μl magnetic beads Axygen beads, and mix with a pipette;

[0108] b) Place the sample on the magnetic separation rack until the supernatant is clear, then pipette the supernatant into another 1.5ml low adsorption tube;

[0109] c) Add 65 μl magnetic beads Axygen beads to resuspend with a pipette and mix well, place on a magnetic separation rack until the supernatant is clear, discard the supernatant, add 50 μl of 75% ethanol to wash, remove the residual liquid with the pipette, and dry in the air;

[0110] d) Add 56 μL of enzyme-free water, mix well, pipette 51 μl of clarified liquid into a new 1.5ml centrifuge tube;

[0111] e) Take 1 μl to measure DNA concentration with Qubit. The resulting 50 μl ctDNA small fragments were directly used in the cfDNA KAPA library construct...

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Abstract

The invention relates to a method for extracting cfDNA (cell-free deoxyribonucleic acid) from a hydrothorax sample to construct a high-throughput sequencing library, and belongs to the technical field of gene detection. The invention successfully extracts the cfDNA from hydrothorax for the first time; and after verification, the cfDNA can be used as a sample source for constructing a high-throughput sequencing library and can become an effective supplement to plasma cfDNA. Besides, large and small segments of the cfDNA in the hydrothorax sample are separated according to the method, so that the cfDNA in the hydrothorax sample is higher in purity, thereby being more suitable for DNA library construction, being more beneficial to later period mutation detection and being capable of solving the problems involving sample singleness and detection rate in conventional simplex plasma based cfDNA extraction and library construction.

Description

technical field [0001] The invention relates to a method for extracting cfDNA from pleural fluid, a kit and a constructed cfDNA library, belonging to the technical field of gene detection. Background technique [0002] With increasing morbidity and mortality, cancer has become the leading cause of death and a major public health problem in China. According to statistics from the National Cancer Center, there were about 4.29 million new cancer cases and 2.81 million cancer deaths in China in 2015. Lung cancer became the most common cancer and the leading cause of cancer death. With the continuous rise of various treatment methods, such as early radiotherapy, modern chemotherapy, and current targeted therapy and immunotherapy, cancer treatment is gradually developing towards a precise treatment model, especially targeted therapy and immunotherapy. This is particularly important for the screening of specific target populations, and as an indispensable clinical auxiliary mean...

Claims

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Application Information

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IPC IPC(8): C12N15/10C40B40/06C40B50/06C12Q1/68
CPCC12N15/101C12N15/1093C12Q1/6806C12Q1/6869C40B40/06C40B50/06C12Q2523/308C12Q2527/125C12Q2525/191C12Q2563/143C12Q2563/149C12Q2535/122
Inventor 邵阳汪笑男吴雪王富锋包华刘一川
Owner NANJING SHIHE MEDICAL DEVICES CO LTD
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