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 su

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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[0042] Example 1,

[0043] The formula of the phlegm solution is determined: 1) It is convenient for clinical and laboratory acquisition; 2) It must be an isotonic liquid to ensure that the sputum cytology is intact and does not affect the cytological pathological diagnosis; 3) Minimize the phlegm solution in the supernatant The salt ion content of the supernatant avoids affecting the extraction efficiency of the cfDNA kit extracted from the supernatant; 4) Dithiothreitol and β-mercaptoethanol are commonly used denaturants in experiments, but dithiothreitol is colorless and tasteless and more suitable for laboratories Use; 5) The literature reports that there is a big difference in the concentration of DTT to resolve phlegm [1]. Under different salt ion concentrations, the optimal concentration of dithiothreitol requires experimental proof.

[0044] In order to observe whether the concentration of dithiothreitol changes the phlegm-reducing effect, this experiment was conducted with...

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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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