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Phlegm resolving liquid for extracting free DNAs from cell-free supernatant of sputum specimen

A cell-free and sputum technology, applied in the field of sputum removal, can solve the problems of low cfDNA content in cell-free supernatant, affecting the accuracy of gene detection results, etc., and achieve the effects of abundant cfDNA content, easy availability, and widespread promotion.

Active Publication Date: 2019-05-17
BEIJING HOSPITAL
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Problems solved by technology

However, because this kind of sputum solution contains methanol or other unknown salts, the amount of cfDNA in the cell-free supernatant extracted after dissolving the sputum sample is low, which affects the accuracy of subsequent genetic testing results.

Method used

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  • Phlegm resolving liquid for extracting free DNAs from cell-free supernatant of sputum specimen
  • Phlegm resolving liquid for extracting free DNAs from cell-free supernatant of sputum specimen
  • Phlegm resolving liquid for extracting free DNAs from cell-free supernatant of sputum specimen

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Embodiment 1

[0043] Determination of the formula of phlegm-resolving liquid: 1) It is easy to obtain clinically and in the laboratory; 2) It must be an isotonic liquid to ensure that the cytology of the sputum is intact and does not affect the cytopathological diagnosis; 4) Dithiothreitol and β-mercaptoethanol are commonly used denaturants in experiments, but dithiothreitol is colorless and tasteless and is more suitable for laboratories Use; 5) According to literature reports, there is a large difference in the concentration of DTT for reducing phlegm [1]. Under different salt ion concentrations, the optimal concentration of dithiothreitol for reducing phlegm needs to be proved experimentally.

[0044] In order to observe whether the concentration of dithiothreitol changes the effect of reducing phlegm, this experiment was observed from different dithiothreitol concentrations of 0.1mol / L, 0.3mol / L, 0.5mol / L, and 0.7mol / L mol / L , from the two aspects of phlegm-resolving time and phlegm-res...

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Abstract

The invention provides a phlegm resolving liquid for extracting free DNAs from a cell-free supernatant of a sputum specimen. The phlegm resolving liquid consists of physiological saline and a dithiothreitol water solution with the concentration of 0.1-0.7 mol / L; the physiological saline and the dithiothreitol water solution are independently packaged and stored before use; normal saline is mixed with the sputum specimen and the dithiothreitol water solution on the use site. The volume ratio of the physiological saline to the dithiothreitol water solution is 9:1. Reagents of a formula are easily obtained, a preparation method is simple, and wide popularization is promoted. Compared with a phlegm resolving liquid reagent which has been applied clinically, the phlegm resolving liquid has advantages in the aspects of the phlegm resolving effect, the amount of obtained cfDNAs and the accuracy of a gene change detection result. Since the amount of the obtained cfDNAs is large, different kinds of genes can be detected. The formula belongs to isotonic liquids; after the cell-free supernatant is collected through short-term treatment, a cell fixing liquid is added into a precipitate, correspondingly cell morphology cannot be damaged, and a subsequent cytological pathological diagnosis result is not influenced.

Description

technical field [0001] The present invention relates to a phlegm-resolving liquid for extracting free DNA in the cell-free supernatant of a sputum sample, and in particular to a phlegm-resolving liquid formula for extracting non-small cell lung cancer (non-small cell lung cancer, NSCLC) patients The sputum sample has no free DNA (cell free DNA, cfDNA) in the supernatant, and the obtained cfDNA can be used for detection of relevant driver gene changes in NSCLC patients. Background technique [0002] In recent years, the role of targeted drugs in the clinical tumor treatment of NSCLC has been confirmed, which is the so-called "precision treatment". The application of targeted drug therapy requires different gene change detection to select patients, and at the same time, corresponding drug selection is carried out for different gene changes carried. After tumor progression in patients resistant to targeted drug therapy, genetic alteration detection is still required to guide s...

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杨丽李琳李晓光李元明焦鹏阮力宋庆涛
Owner BEIJING HOSPITAL
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