Sputum liquefaction reagent, kit containing same and application of kit

A technology of sputum and reagents, which is applied in the field of life sciences, can solve the problems of no change in the ratio of human genome DNA, large loss of cells and pathogenic bacteria, and easy clogging of microfluidic chips, so as to promote liquefaction and reduce human cell genome. DNA content, the effect of meeting the experimental operation requirements

Inactive Publication Date: 2018-08-17
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, the methods used for sputum liquefaction mainly include protease method, reduction method (DTT and acetylcysteine) and alkali lysis method. The loss of pathogenic bacteria is large. The DTT method is suitable for cytological detection and is better for retaining cells. However, there are also too many static sediments after liquefaction. These filamentous or granular sediments under the microscope are easy to block the microfluidics. Micron-scale equipment such as chips, resulting in too little sample volume and collected liquid volume, which cannot meet the experimental requirements
In addition, DNA released from necrotic cells cannot be dissociated in the supernatant because it is wrapped by histones and mucins, and will precipitate with the bacteria during centrifugation, resulting in the presence of a large amount of human genomic DNA in the isolated bacteria
The experimental results of our patented CN107090399A fluidic chip to separate bacteria from sputum further proved this point: there were no Hoechst 33342-stained cells and nuclei in the side channel collection liquid under a fluorescence microscope, but the ratio of bacterial genomic DNA to human genomic DNA varied greatly. There is little or no change, which indicates that there is a large amount of genomic DNA released after cell necrosis wrapped in protein complexes and precipitated with centrifugation instead of being free in the supernatant. Secondly, bacteria may also be attached or entrapped in filaments The protein is not fully released and flows away with the liquid flow in the main channel, resulting in a decrease in the amount of bacteria collected

Method used

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  • Sputum liquefaction reagent, kit containing same and application of kit
  • Sputum liquefaction reagent, kit containing same and application of kit
  • Sputum liquefaction reagent, kit containing same and application of kit

Examples

Experimental program
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Effect test

Embodiment 1

[0034] The sputum liquefaction reagent includes fixative, liquefaction agent, glass beads, exonuclease and buffer, and the kit composed of the sputum liquefaction reagent includes fixative, liquefaction agent, glass beads, exonuclease and buffer, 40μm filter screen six components.

[0035] The specific composition of each component is as follows:

[0036] Fixative: 50% ethanol solution (volume percentage), 100 μM sodium polyaspartate;

[0037] 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;

[0038] Glass beads: 1-5mm in diameter, ultrasonically cleaned once in sterile deionized water, washed 2-3 times in sterile deionized water, and autoclaved;

[0039] Exonuclease and buffer: DNase I recombinant 2,000units / ml (NEB Company); exonuclease 2×B...

Embodiment 2

[0041] Embodiment 2: sputum liquefaction process

[0042] Use embodiment 1 sputum liquefaction reagent.

[0043] Transfer the sputum to a 50ml sterile centrifuge tube, add 1 times the volume of fixative and 1 times the volume of liquefaction agent relative to the volume of sputum, add glass beads accounting for 2 / 3 of the volume of the mixture to the mixture, and place it horizontally on Vertical rotation mixer (Shanghai Kehuai Instruments), vertical rotation at room temperature, rotation speed 20r / min, liquefaction for 30 minutes. Then add 2×DNAase I enzyme buffer and enzyme equal to the volume of the liquid (the volume of each liquid is: 1mL sputum, 1mL fixative, 1mL liquefaction agent, 3mL DNAase I enzyme buffer, add 10μL DNAase to each mL enzyme buffer 1 enzyme), after standing at 37°C for 30min, observe that there is no adhesion of protein fibers on the tube wall and no lumps in the liquefied solution, filter through a 40 μm filter screen, and it is qualified if there is...

Embodiment 3

[0047] Take 1 mL of the sputum liquefaction solution after liquefaction by the method of Example 2, and use a microfluidic chip (patent CN107090399A) to purify the bacteria in the liquefied sputum sample, and the chip throughput of the mixed suspension of cultured cells and bacteria reaches 450 microliters ( figure 2 of Vn). The volume of the collected samples after purification of the sputum and the comparative analysis of the colonies on the blood plate were analyzed. Dilute the collection solution 10 and 100 times, take 10 μl of it and apply it on a blood plate under sterile conditions, culture overnight at 37°C, and count the clones.

[0048] see figure 2 and image 3 . It can be seen that the liquefaction reagent has improved the liquefaction efficiency and the bacterial release efficiency, and the volume of the collected liquid and the number of colony clones are about 3 times and 5 times of the volume of the collected liquid obtained by the conventional DTT method....

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Abstract

The invention provides a sputum liquefaction reagent which comprises a fixing agent, liquefaction liquid, glass beads and exonuclease buffer solution. The fixing agent contains a solvent and a polymer, the polymer is sodium polyaspartate or poly-L-glutamic acid, and the liquefaction liquid contains a reducing agent and the buffer solution with the pH (potential of hydrogen) value of 7.0-8.5. The invention further provides a kit containing the sputum liquefaction reagent and an application of the kit. According to the sputum liquefaction reagent, sputum is liquefied from the aspects such as retention of integrity of cells and bacteria, improving of sputum liquefaction efficiency and removal of a macromolecule DNA (deoxyribonucleic acid) compound, and liquefied sputum can effectively meet bacterium separation of a micro-sized micro-fluidic chip. The reagent has the advantages that liquefaction time can be shortened, liquefaction efficiency is improved, bacterium separation efficiency isimproved, and the DNA content of a human cell genome is reduced.

Description

technical field [0001] The invention belongs to the field of life sciences, and in particular relates to the preparation and application of a reagent or kit of a sputum liquefaction reagent. 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28
CPCG01N1/28
Inventor 邵长君康禹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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