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Method for extracting cfDNA in sputum sample

A sputum and specimen technology, which is applied in the field of cell-free DNA extraction from cell-free supernatant of sputum specimens, can solve the problems of low diagnostic sensitivity and low proportion of cancer cells.

Active Publication Date: 2019-05-17
BEIJING HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] However, the application of tumor cells in sputum samples to detect gene changes also has problems, so it has always been considered by the industry as a difficult sample for gene detection. First, the proportion of cancer cells found in sputum samples is not high (only 30%), and the diagnostic sensitivity is low; Second, even if cancer cells are found, they are almost all samples with low tumor cell content, and only a few sputum samples can be detected by enriching cancer cells in sputum smears

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  • Method for extracting cfDNA in sputum sample
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  • Method for extracting cfDNA in sputum sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The present invention provides a method for extracting cfDNA from sputum samples, which includes the following steps:

[0046] Collection of sputum specimens: First, select NSCLC patients who are suitable for the application of sputum specimens for genetic alteration testing: patients with advanced (excluding locally advanced) NSCLC, and patients with natural discharge of sputum (no need to induce sputum by nebulization); secondly , Collection of sputum: sputum specimens of patients with advanced NSCLC (sputum specimens that are naturally coughed up without induction), the amount of sputum specimens collected is 1-3ml, if the amount of sputum is not enough, it can be collected in batches, and the sputum specimens are placed in the collection process When the sputum sample reaches a sufficient amount at -20°C, proceed to the next step; it is recommended that the sputum sample be transported at a temperature of 2-8°C during transportation.

[0047] Storage of sputum specimens:...

Embodiment 2

[0055] In the phlegm-resolving step in Example 1, use elbow tweezers to pick 1ml specimens from the collected sputum (try to pick out blood-stained sputum specimens), place them in a 50ml centrifuge tube, and add 10ml sputum-resolving liquid ( First add 9ml of normal saline, and then add 1ml of 0.5mol / L dithiothreitol aqueous solution)" to "Use elbow tweezers to pick 1.5ml specimens from the collected sputum (try to pick bloodshot sputum specimens), Place it in a 50ml centrifuge tube, add 8ml of phlegm solution (first add 7ml of physiological saline, and then add 1ml of 0.3mol / L dithiothreitol aqueous solution). The rest are the same as the method in Example 1.

[0056] At this time, 10 cases of 2 kinds of paired sputum cell-free supernatant specimens (Comparison of the different proportions of the sputum solution in Example 2 and Example 1, the concentration of cfDNA extracted in Example 1 (1.99-24ng / μl), and Example 2 The concentration of cfDNA extracted from CF (0.89-27ng / μl),...

Embodiment 3

[0058] In the phlegm-resolving step in Example 1, use elbow tweezers to pick 1ml specimens from the collected sputum (try to pick out blood-stained sputum specimens), place them in a 50ml centrifuge tube, and add 10ml sputum-resolving liquid ( First add 9ml of normal saline, and then add 1ml of 0.5mol / L dithiothreitol aqueous solution)" to "Use elbow tweezers to pick 2ml specimens from the collected sputum (try to pick bloodshot sputum specimens), and place In a 50ml centrifuge tube, add 12ml of phlegm solution (first add 11ml of physiological saline, and then add 1ml of 0.7mol / L DTT aqueous solution). The rest are the same as in Example 1.

[0059] At this time, 10 cases of 2 kinds of paired sputum cell-free supernatant sample formulations (Example 3 and Example 1 phlegm solution different ratio formulations compared the same amount of sputum samples, the concentration of cfDNA extracted in Example 1 (1.99-24ng / μl) ), while the concentration of cfDNA extracted in Example 3 (1.01...

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Abstract

The invention relates to the field of DNA extraction methods, and provides a method for extracting cfDNA in a sputum sample, which comprises the steps of preparing an extraction sample of cfDNA in sputum and extracting the cfDNA in the extraction sample by using a kit, The preparation method of the cfDNA extraction sample in the sputum comprises the following steps of: 1) collecting sputum; 2) controlling the quality of the sputum sample; 3) mixing the sputum sample with phlegm-eliminating liquid, and shaking until the phlegm is completely eliminated to obtain a phlegm-eliminating mixed solution; 4) carrying out centrifugal treatment on the phlegm-eliminating mixed solution, and separating supernatant and bottom precipitate, wherein the supernatant is cell-free supernatant; 5) centrifugingthe cell-free supernatant, and collecting the supernatant to obtain the extraction sample. The method for extracting the cfDNA in the sputum sample provides a standardized extraction flow of the cfDNA in the sputum sample, and can obtain enough cfDNA from the sputum sample to provide a basis for subsequent gene change detection.

Description

Technical field [0001] The invention relates to a method for extracting DNA, in particular to a method for extracting free DNA (Cell Free DNA, cfDNA) in the cell-free supernatant of a sputum sample. Background technique [0002] In recent years, the application of targeted drugs for the treatment of non-small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC) has become the standard treatment method in clinical tumor treatment, the so-called "precision treatment". The application of targeted drug therapy requires different genetic change detection, and the corresponding drug selection is carried out for the different genetic changes carried. After targeted drug treatment of resistant patients with tumor progression, genetic change testing is still needed to guide subsequent treatment, so genetic change testing runs through the entire process of NSCLC treatment. Both domestic and foreign NSCLC diagnosis and treatment guidelines recommend that patients routinely undergo a set of...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806
Inventor 王征刘东戈张琳施红孙明君崔娣李琳徐燕王孟昭邸婧阮力宋庆涛
Owner BEIJING HOSPITAL
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