Extraction method of cfdna in sputum specimen

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

Active Publication Date: 2021-06-29
BEIJING HOSPITAL
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  • Abstract
  • Description
  • Claims
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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

Method used

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  • Extraction method of cfdna in sputum specimen
  • Extraction method of cfdna in sputum specimen
  • Extraction method of cfdna in sputum specimen

Examples

Experimental program
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Embodiment 1

[0045] The invention provides a method for extracting cfDNA in a sputum sample, comprising the following steps:

[0046] Collection of sputum specimens: First, select NSCLC patients who are suitable for genetic alteration detection using sputum specimens: advanced (excluding locally advanced) NSCLC patients, and patients who can naturally excrete sputum (nebulization is not required to induce sputum); secondly , Collect sputum: sputum samples from patients with advanced NSCLC (sputum samples are naturally coughed up without induction), and the amount of sputum samples collected is 1-3ml. If the amount of sputum is not enough, it can be collected in stages. -20°C, after the sputum sample reaches a sufficient amount, proceed to the next step; it is recommended to keep the sputum sample at a temperature of 2-8°C during the transportation process.

[0047] Storage of sputum specimens: After sending to the testing center, it is recommended to immediately reduce the sputum, collect ...

Embodiment 2

[0055] In the step of reducing phlegm in Example 1, "use elbow tweezers to pick 1ml of specimen from the collected sputum (try to pick blood-streaked sputum specimen), place it in a 50ml centrifuge tube, add 10ml of sputum-resolving ( First add 9 ml of normal saline, then add 1 ml of 0.5 mol / L dithiothreitol aqueous solution)” to “Pick 1.5 ml of specimens from the collected sputum with curved tweezers (try to pick blood-streaked sputum specimens), Place in a 50ml centrifuge tube, add 8ml of sputum-resolving liquid (add 7ml of normal saline first, then add 1ml of 0.3mol / L dithiothreitol aqueous solution)" and 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 samples (comparison of 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 the concentration of cfDNA in Example 2 The cfDNA concentration (0.89-27ng / μl...

Embodiment 3

[0058] In the step of reducing phlegm in Example 1, "use elbow tweezers to pick 1ml of specimen from the collected sputum (try to pick blood-streaked sputum specimen), place it in a 50ml centrifuge tube, add 10ml of sputum-resolving ( First add 9 ml of normal saline, then add 1 ml of 0.5 mol / L dithiothreitol aqueous solution)” to “Pick 2 ml of specimens from the collected sputum with curved tweezers (try to pick blood-streaked sputum specimens), set In the 50ml centrifuge tube, add 12ml of sputum-resolving liquid (add 11ml of normal saline first, then add 1ml of 0.7mol / L DTT aqueous solution)" and the rest are all the same as in Example 1.

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

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Abstract

The present invention relates to the field of DNA extraction methods, and provides a method for extracting cfDNA in sputum samples, which includes preparing cfDNA extraction samples in sputum and using a kit for extracting cfDNA to extract cfDNA from the samples. The preparation method of cfDNA extraction sample in liquid comprises the following steps: 1) sputum collection; 2) quality control of sputum sample; 3) mixing sputum sample with phlegm-resolving liquid, shaking until phlegm-resolving is complete, and obtaining mixed phlegm-resolving 4) Centrifuge the phlegm-resolving mixture, separate the supernatant and the bottom sediment, and the supernatant is the cell-free supernatant; 5) Centrifuge the cell-free supernatant and collect the supernatant , to get the extracted sample. The method for extracting cfDNA in a sputum sample of the present invention provides a standardized cfDNA extraction process in a sputum sample, and can obtain a sufficient amount of 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 to treat non-small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC) has become a standard treatment method in clinical tumor treatment, which is the so-called "precision therapy". The application of targeted drug therapy requires different gene change detection, and corresponding drug selection is carried out according to the different gene changes carried. After tumor progression in patients resistant to targeted drug therapy, genetic alteration detection is still required to guide subsequent treatment. Therefore, genetic alteration detection runs through the entire process of NSCLC treatment. Domestic and foreign NSCLC diagnosis and treatment guidelines recommend that patients routinely underg...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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