Rapid purification reagent for sputum microorganisms, and application of rapid purification reagent

A technology of microorganisms and sputum, applied in the field of life sciences, can solve the problems of low sample efficiency, no detection, and uneven enrichment effect of bacteria, and achieve the effect of simple and fast operation, good adaptability, and improved bacterial enrichment efficiency

Active Publication Date: 2018-11-13
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Grade 2: The appearance of sputum is thicker than that of Grade 1. After suction, a small amount of sputum remains on the inner wall of the glass joint, but it is easily washed away by water. Grade 3: The appearance of sputum is obviously viscous and yellow. The pressure is too large and collapses, and a large amount of sputum often stays on the inner wall of the glass joint and is not easy to be washed away by water
[0006] At present, there are few chemical methods and reagents for enriching sputum microorganisms and removing human cell genomic DNA. The existing method of enriching microorganisms by lysing cells through hypotonic methods is uneven in enriching bacteria, and most samples are inefficient. Moreover, it is found in oral swabs and saliva samples, and a large amount of high-molecular mucin in sputum is difficult to dissolve in water. Therefore, there is no report on the use of this method for microbial enrichment in sputum.

Method used

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  • Rapid purification reagent for sputum microorganisms, and application of rapid purification reagent
  • Rapid purification reagent for sputum microorganisms, and application of rapid purification reagent
  • Rapid purification reagent for sputum microorganisms, and application of rapid purification reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Cell (nuclear) lysate component optimization test

[0032] High-concentration guanidine hydrochloride (6M) is a strong denaturant of proteins. Use low-concentration guanidine hydrochloride (less than 1M) to culture cells and observe the disintegration of cells.

[0033] Take the culture flask of H460 epithelial cells just full, pour out the culture medium, add 1M guanidine hydrochloride and 0.5M guanidine hydrochloride, and 1M guanidine hydrochloride + 0.03% IGEPAL-CA630, 0.5M + 0.03% IGEPAL-CA630, 0.5M + 0.03% IGEPAL- The four experimental groups of CA630 guanidine hydrochloride were observed by time. The cells in the guanidine hydrochloride group did not disintegrate within 20 minutes, but the cells in the group mixed with IGEPAL-CA630 disintegrated soon. This experiment shows that the combination of guanidine hydrochloride and surfactant can lyse cells more effectively.

[0034] On the basis of this test, the volume concentration of IGEPAL-CA630 in the...

Embodiment 2

[0036] The rapid purification reagent of sputum and sputum microorganisms comprises liquefier, cell lysate and exonuclease buffer.

[0037] The specific composition of each component is as follows:

[0038] Liquefaction agent: 0.1g DTT (molecular weight 154.25), 0.78g sodium chloride, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, 0.112g sodium dihydrogen phosphate, adjust the pH value to 7.6 to 8.0 with 1M NaOH, add dihydrogen Distill water to 100mL, filter to sterilize;

[0039] Cell lysate: prepared with double distilled water, containing 2M guanidine hydrochloride, 0.06% (volume fraction) IGEPAL-CA630, filtered to sterilize;

[0040] Exonuclease and buffer: DNase I recombinant 2,000units / mL (NEB Company); exonuclease 2×Buffer: 20mM Tris-HCl, 5mM MgCl 2 , 1mM CaCl 2 , pH 7.6;

Embodiment 3

[0041] Example 3: Reagents for rapid purification of sputum microorganisms

[0042] The reagent for rapid purification of sputum microorganisms in Example 2 was used.

[0043]Transfer 1 volume of sputum to a 50ml sterile centrifuge tube, add a volume of liquefier that is 1 times the volume of sputum, and shake for 10 minutes to liquefy. Divide the equal parts into two parts, one part is used as the control (control group), add an equal volume of cell lysate (the same volume as (sputum + liquefier)) to the other part (experimental group), and lyse for 5-15 minutes After the sputum was observed to be a clear water sample, the bacteria were collected by centrifugation, the supernatant was removed, and 200 μL of DNAase I enzyme buffer and enzyme were added to the collected bacteria (the liquid volume ratio was: 1 mL of sputum, 200 μL of DNAase I enzyme buffer, as per mL enzyme buffer plus 10 μL DNAase I enzyme), let stand at 37°C for 30 min. (DNAase I enzyme can be inactivated a...

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Abstract

The invention provides a rapid purification reagent for sputum microorganisms. The rapid purification reagent is prepared from a liquefaction solution, cell lysate and an exonuclease buffer solution,wherein the cell lysate contains guanidine salt and a surface active agent, and the concentration of the guanidine salt in the cell lysate is 1-3mol/L. The invention also provides application of the rapid purification reagent for the sputum microorganisms. The rapid purification reagent, provided by the invention, for the sputum microorganisms is prepared by combining low-concentration guanidine hydrochloride with the surface active agent and a reducing agent for completely lysing human cells (nucleus), and genomic DNA is released into a solution due to cell disintegration, most of the DNA isremoved during centrifugal obtaining of bacteria, and the residual free DNA is further digested by DNase; the bacteria are not affected or seldom affected due to the protection of the cell wall.

Description

technical field [0001] The invention belongs to the field of life sciences, and in particular relates to a microorganism purification reagent and its application. Background technique [0002] The fluid in sputum is mainly secreted by mucus-secreting glands and goblet cells of the bronchial mucosal epithelium. Under normal circumstances, goblet cells and glands secrete a small amount of mucus to cover the surface of the mucous membrane, which can protect the mucous membrane and keep the tracheal mucous membrane moist, so as to adhere to the dust particles and bacteria inhaled into the trachea and bronchi, and block them. It enters deep into the lung tissue, and then, with the help of the ciliary swing of the ciliated columnar epithelium, it is expelled to the larynx at the upper end of the trachea, and is spit out through the oral cavity. Normal sputum is generally colorless and transparent, a little viscous, and slightly viscous. An appropriate amount of sputum helps to mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 邵长君康禹石兴于军林强王建楚亚男付荣荣
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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