CN108414313A - A kind of sputum liquefaction agent, the kit containing it and its application - Google Patents
A kind of sputum liquefaction agent, the kit containing it and its application Download PDFInfo
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- CN108414313A CN108414313A CN201810161127.XA CN201810161127A CN108414313A CN 108414313 A CN108414313 A CN 108414313A CN 201810161127 A CN201810161127 A CN 201810161127A CN 108414313 A CN108414313 A CN 108414313A
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
The present invention proposes a kind of sputum liquefaction agent, including fixative, liquefier, bead, Exonucleolytic enzyme buffer liquid；Contain solvent and polymer in the fixative, the polymer is poly (sodium aspartate) or poly- L glutamic acid, the buffer solution for being 7.0~8.5 containing reducing agent and pH value in the liquefier.The present invention also proposes the kit containing the sputum liquefaction agent and its application.The sputum liquefaction agent of the present invention is started with from reservation cell and bacterium integrality, raising sputum liquefaction efficiency and elimination macromolecule DNA compounds etc. liquefies to sputum, and the separation of bacterial that the sputum after liquefaction can preferably meet micron order micro-fluidic chip uses.The reagent advantage of the present invention is can to shorten liquefying time, improve liquefaction efficiency, improves bacterium separative efficiency, reduces people's cell Genome DNA content.
Description
Technical field
The invention belongs to life sciences, and in particular to a kind of reagent of sputum liquefaction agent or the preparation of kit and
Using.
Background technology
Liquid is mainly to be secreted by the muciparous body of gland and goblet cell of tunica mucosa bronchiorum epithelium in sputum.Normally
In the case of, goblet cell and a small amount of mucus of glandular secretion are covered in mucous membrane layer surface, shield to mucous membrane, can keep tracheae
The moistening of mucous membrane stops that it enters lung tissue depths so that dust granules, the bacterium etc. in sucking trachea-bronchial epithelial cell are adhered to,
Then, then by means of ciliated columnar epithelium fibre swing, they are discharged to the larynx position of tracheae upper end, direct oral cavity is brought up.
Normal phlegm is generally water white transparency, and band slightly viscous is slightly sticky, and suitable sputum helps to moisten respiratory tract, captures dust
With microorganism (mucus).Although any expectoration phlegm can be considered as abnormal, change in no any respiratory system pathology
It also can expectoration or a small amount of phlegm that spues under change.However, in some cases, especially related with respiratory inflammation, amount of expectoration may
It will produce excessive.In these pathology cases, the color of phlegm, quality even smell may all change.
When trachea-bronchial epithelial cell and lung are inflamed by the stimulation or pathogenic infection of adverse factor, respiratory tract
Mucous hyperemia, oedema, massive inflammatory cells infiltrated, blood vessel dilatation, exudation increase, the goblet cell of mucous layer and submucosa
Glandular hyperplasia is loose, and mucous secretion largely increases, and is conducive to remove foreign matter.Polyblennia has just aggravated on ciliated columnar
The burden of skin is unfavorable for the discharge of mucus, under the action of bacterium and its toxin, generates some degeneration necrosis histocytes, pool
It stays in bronchus, the histocyte of mucus and these degeneration necrosis just constitutes phlegm, and more next than normal phlegm there are two features：When
Viscosity higher, second is that color is also possible to become yellow, green, rust or brown from white or grey, it may also also be because
Containing a small amount of blood, to there is trace of blood presentation.
Can be simply divided into phlegm according to the viscosity increase of sputum has foam (serosity) phlegm, mucous sputum and purulence
Phlegm.Clinically the character according to sputum during suction sputum at sputum aspirator tube adapter glass and the adhesion condition in glass inside pipe wall will
The viscosity of sputum is divided into three degree：1 degree：Sputum such as thin rice gruel or foam sample are detained on adapter glass inner wall without sputum after suction sputum.2
Degree：The appearance of phlegm is sticky compared with 1 degree, has a small amount of sputum to be detained in adapter glass inner wall after suction sputum, but is easy to wash down 3 degree by water：Phlegm
Appearance it is obviously sticky, be in yellow, sputum aspirator tube often collapses because negative pressure is excessive, be often detained on adapter glass inner wall a large amount of sputums and
It is not easy to be washed down by water.
The viscosity of sputum is mainly since the acid glycoprotein content in phlegm is related, since glycoprotein molecule is by different
Key (such as disulfide bond, hydrogen bond) cross-join together, form a kind of gel net.Ca in sputum2+Content is high, can increase
Viscosity, microscopically observation to long fibre be exactly to be made of these mucopolysaccharides, mucin.When respiratory tract infection, due to big
The DNA that the core for measuring inflammatory cell is destroyed and generated is wrapped up by histone and mucoprotein, and the viscosity of sputum can also significantly improved,
Purulent sputum is seen, is in often thick parallel fibers.
Include mainly protease method, reduction method (DTT and acetylcysteine) and alkali cracking currently used for sputum liquefaction method
Solution, these methods all deposit certain disadvantage, and wherein protease method and alkaline lysis leads to cell and pathogen damage in sputum
Lose it is larger, DTT methods suitable for cytology detect, to retain cell it is preferable, but there is also liquefaction after quiescent setting object it is excessive,
Filamentous or granular precipitation is presented under these microscopes to be easy to easily block the micron orders equipment such as micro-fluidic chip, so as to cause
Loading volume and collection hypovolia, cannot meet requirement of experiment.In addition, the DNA of non-viable non-apoptotic cell release due to by histone and
Mucoprotein is wrapped up and cannot be free in supernatant, and when centrifugation can get off with bacterium coprecipitation, and separation is caused to obtain in bacterium
The presence of also a large amount of human gene group DNAs.We detach the reality of bacterium in sputum using patent CN107090399A fluidic chips
It tests result and has also further demonstrated that this point：There is no the dyeing of Hoechst 33342 in fluorescence microscope wing passage collection liquid
Cell and nucleus, but bacterial genomes DNA and human gene group DNA's ratio are varied less even without variation, this shows to deposit
The genomic DNA discharged after a large amount of meronecrosis is wrapped in albumen composition, precipitates rather than swims with centrifugation
From in supernatant, secondly bacterium is also possible to fail fully to discharge in silk-like proteins in adhering to or wrap, in main channel with liquid stream
It flows away, bacterium collecting amount is caused to reduce.
Invention content
In view of the deficiencies of the prior art, the purpose of the present invention is to propose to a kind of sputum liquefaction agents.
Second object of the present invention is to propose the kit containing the sputum liquefaction agent.
Third object of the present invention is to propose the application of the sputum liquefaction agent.
Realize that the technical solution of the object of the invention is：
A kind of sputum liquefaction agent, including fixative, liquefier, bead, exonuclease and Exonucleolytic enzyme buffer
Liquid；Contain solvent and polymer in the fixative, the polymer is poly (sodium aspartate) or Poly-L-glutamic acid；The liquid
Change the buffer solution for being 7.0~8.5 containing reducing agent and pH value in liquid, the reducing agent is dithiothreitol (DTT), three (2- carboxylic second
Base) phosphonium salt hydrochlorate, one kind in dithioerythritol.
Wherein, the solvent in the fixative is the ethanol solution of volumetric concentration 40~60%, and poly-aspartate is described
The molecular weight of a concentration of 10~1000 μm of ol/L in solvent, the poly (sodium aspartate) are 2000~50000；The poly- L-
A concentration of 100~1000 μm ol/L of the glutamic acid in the solvent, molecular weight are 5000~1,500,000.
Wherein, the buffer solution for being 7.6~8.0 containing reducing agent and pH value in the liquefier, the buffer solution are
Phosphate or NaHCO3Buffer solution；The reducing agent is selected from dithiothreitol (DTT), the molar concentration 5- of 0.05~0.2g/100mL
One kind in three (2- carboxyethyls) phosphonium salt hydrochlorates of 200mmol/L, the dithioerythritol of 0.05~0.2g/100mL.
Wherein, a diameter of 1~5mm of the bead.
Wherein, contain 0~20 unit exonuclease in every mL Exonucleolytic enzyme buffer liquids.The exonuclease is
recombinant DNase I。
Preferably, the sputum liquefaction agent includes 1~3 part of 1~3 part of fixative, liquefier, the bead of parts by volume
2~6 parts, 3~8 parts of Exonucleolytic enzyme buffer liquid.
Parts by volume of the present invention refers to the common volume unit of those skilled in the art, for example, can be milliliter, microlitre,
Ounce or for the easy to operate volume unit made by oneself.In the same formula, the unit volume that " part " indicates is identical.
A kind of sputum liquefaction agent box, including sputum liquefaction agent of the present invention and aseptic filtration net, the nothing
The aperture of bacterium filter screen is 40~100 μm.
Application of sputum liquefaction agent or the sputum liquefaction agent box of the present invention in sputum liquefaction.
Using the method that the sputum liquefaction agent carries out sputum liquefaction, including operation：
1) sputum that volume is 0.8~1.2 times of fixative is transferred in container, fixer and liquefier is added, then add
Enter bead, liquefies 15~30 minutes；
2) Exonucleolytic enzyme buffer liquid is added, observes on chamber wall without being deposited without agglomerate in the adherency of azelon silk and liquefier
Using filter screen filtration.
After Exonucleolytic enzyme buffer liquid is added in step 2), 25~37 DEG C of placement 15-30min.
The beneficial effects of the present invention are：
Sputum liquefaction agent proposed by the present invention drives phlegm silk grinding by the way that bead is added, and promotes reducing agent liquefaction effect
Fruit, intuitive effect are that liquefying time is short, not big agglomerate, the volume throughput after same sputum sample liquefaction and wing passage
Collection can improve 3 times or more, substantially meet experimental implementation requirement.Secondly sputum liquefaction agent proposed by the present invention passes through addition
Poly (sodium aspartate) or Poly-L-glutamic acid promote the DNA depolymerization and exposure wrapped up by histone and mucoprotein, with DNase into one
Step digestion, and bacterium due to cell wall be protected from influence or influence very little.In conclusion this liquefied reagent passes through enhancing
Liquefaction effect is reduced and is blocked, and improves collected volume, and the strategies such as DNA of degradation of cell release account for improve bacterial genomes DNA
Than.The two generation DNA sequencing results that we test also indicate that the liquefied reagent using the present invention, micro- using patent CN107090399A
Bacterial genomes accounting in the macro genome of patient's COPD sputum can be improved 10 times or more by fluidic chip.
The sputum liquefaction agent of the present invention is reduced micro- from the integrality, the enhancing sputum liquefaction efficiency that retain cell and bacterium
Little particle, improve loading and collected volume, eliminate non-viable non-apoptotic cell release macromolecule DNA compounds etc. start with to sputum into
Row liquefaction, the sputum after liquefaction can meet separation of the micron order micro-fluidic chip to bacterium.
The reagent advantage of the present invention is can to shorten liquefying time, improve liquefaction efficiency, improves bacterium separative efficiency, reduces
People's cell Genome DNA content.
Description of the drawings
Fig. 1：Micro- sem observation liquefies sputum, and figure is on Fig. 1：Sputum smear Rui Shi-Giemsa staining, figure below are micro-
Sem observation sputum liquefaction liquid.
Fig. 2：1mL liquefies sputum in application micro fluidic device (patent CN107090399A) separation compared with conventional DTT methods
The collection liquid volume (unit is μ L) of acquisition, Vn represents this method and obtains collection liquid volume, and Vd represents routine DTT methods and collected
Liquid accumulates.
Fig. 3：The collection liquid obtained is detached in application micro fluidic device (patent CN107090399A) compared with conventional DTT methods
Blood plate colony clone number is analyzed, and Nn represents the clump count that this method meter obtains, and Nd represents the clump count that routine DTT method meters obtain.
Specific implementation mode
The present invention is now illustrated with following embodiment, but is not limited to the scope of the present invention.
The means used in embodiment use the means of this field routine unless otherwise instructed.
Embodiment 1：
This sputum liquefaction agent includes fixative, liquefier, bead, exonuclease and buffer solution, by this sputum liquid
The kit for changing reagent composition includes fixative, liquefier, bead, exonuclease and buffer solution, 40 μm of filter screens six
Component.
The specific ingredient of each component is as follows：
Fixative：50% ethanol solution (percent by volume), 100 μM of poly (sodium aspartate)s；
Liquefier：0.1g DTT (molecular weight 154.25), 0.78g sodium chloride, 0.02g potassium chloride, 0.02g biphosphates
Potassium, 0.112g sodium dihydrogen phosphates, it is 7.6 to 8.0 that 1M NaOH, which are adjusted to pH value, adds distilled water to 100ml, filtration sterilization；
Bead：A diameter of 1-5mm is cleaned by ultrasonic once in aseptic deionized water, and aseptic deionized water is washed 2-3 times, high
Press sterilization treatment；
Exonuclease and buffer solution：DNase I recombinant 2,000units/ml (NEB companies)；Outside nucleic acid
2 × Buffer of enzyme cutting：20mM Tris-HCl, 5mM MgCl2,1mM CaCl2,pH 7.6；
Aseptic filtration net：40 μm (Thermofisher companies).
Embodiment 2：Sputum liquefaction process
Use 1 sputum liquefaction agent of embodiment.
Sputum is transferred in 50ml sterile centrifugation tubes, the fixer and 1 times of body relative to 1 times of volume of sputum volume is added
Long-pending liquefier is added into mixed liquor and accounts for the bead of 2/3 mixeding liquid volume, be placed horizontally at vertical rotary mixed instrument (on
Hai Kehuai instruments), room temperature vertical rotary, rotating speed 20r/min liquefies 30 minutes.Add the 2 × DNAase isometric with liquid
(each volume of fluid is for I enzyme buffer liquids and enzyme：1mL sputums, 1mL fixatives, 1mL liquefiers, 3mL DNAase I enzyme buffers
Liquid adds 10 μ L DNAase I enzymes by every mL enzyme buffer liquids), after standing 30min at 37 DEG C, observe on tube wall without azelon
Exist without agglomerate in silk adherency and liquefier, filter 40 μm of filter screens, is qualification with nothing or minute quantity remnants.(DNAase I enzymes
75 DEG C of 15min inactivations can be set, such as by being inactivated after chip centrifugal enrichment).
Addition Hoechst 33342 dyes 10min after the sputum that liquefies detaches, and 3000rpm centrifuges 3min, and PBS is washed once, and 1/
50 volume PBS hang, and take 1 μ l drops after glass slide, coverslip mounting, liquefaction effect in fluorescence microscopy sputum.
The result is shown in Figure 1 schemes visible sputum azelon silk and cell on Fig. 1.Figure below of Fig. 1, visible sputum under light field
It is liquefied well, has no azelon silk and big particulate matter.The dyeing observations of Hoechst 33342 show that cell is more complete
It is whole, have no the filamental compound that DNA is formed with albumen.
Embodiment 3：
Sputum liquefaction liquid after taking 2 method of 1mL embodiments to liquefy, utilizes micro-fluidic chip (patent CN107090399A)
Bacterium in refined solution resolving sputum liquid sample, culture cell mix the chip throughput of suspension with bacterium up to 450 microlitres (Vn of Fig. 2).
It collects sample after purification to sputum and carries out volume and blood plate colonies comparative analysis.Collection liquid is diluted 10,100 times, respectively
Take even spread and blood plate under 10 μ l aseptic conditions, 37 DEG C of overnight incubations, colony count.
Referring to Fig. 2 and Fig. 3.It can be seen that improving liquefaction efficiency with this liquefied reagent, bacterium release efficiency is improved, collects liquid
Product and colony clone number are 3 times or so and 5 times or so that routine DTT methods obtain collection liquid volume.
Comparative example
This comparative example uses routine DTT methods, liquefies, takes for the phlegm of patients with chronic obstructive pulmonary diseases (COPD)
Sputum liquefaction liquid after 1mL liquefaction detaches bacterium in sputum using patent CN107090399A micro-fluidic chips, and liquefy sputum
Most 500 microlitres by volume of chip observes that the wing passage that bacterium is collected easily is blocked, and bacterium collection liquid volume only has
150 microlitres (Vd of Fig. 2).There is no cell that Hoechst 33342 is dyed and thin in fluorescence microscope wing passage collection liquid
Karyon, but bacterial genomes DNA and human gene group DNA's ratio are varied less even without variation, and this shows that there is a large amount of
The genomic DNA discharged after meronecrosis is wrapped in albumen composition, precipitates rather than is free in supernatant with centrifugation,
Secondly bacterium is also possible to fail fully to discharge in silk-like proteins in adhering to or wrap, and flows away with liquid stream in main channel, causes thin
Bacterium collecting amount is reduced.
Embodiment 4：Sputum liquefaction process
In this sputum liquefaction agent：
Fixative：100 μM of 50% ethanol solution (percent by volume), poly (sodium aspartate)；
Liquefier：0.1g DTT, 0.78g sodium chloride, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphates, 0.112g di(2-ethylhexyl)phosphates
Hydrogen sodium, it is 7.6 to 8.0 that 1M NaOH, which are adjusted to pH value, adds distilled water to 100ml, filtration sterilization；
Bead：A diameter of 1~5mm, 180 DEG C of processing 4 hours or more.
Other compositions are the same as embodiment 1.
Sputum is transferred in 50ml sterile centrifugation tubes, fixer, liquefier is added, is added into solution and accounts for 2/3 liquid
The bead of volume, is placed horizontally at vertical rotary mixed instrument, room temperature vertical rotary, and rotating speed 20r/min liquefies 15-30 minutes.
2 × DNAase I enzyme buffer liquids (adding 10 μ l DNAase I enzymes by every 1mL buffer solutions) are added, 30min is stood at 37 DEG C
Afterwards, it observes on tube wall without existing without agglomerate in the adherency of azelon silk and liquefier, 40 μm of filter screens is filtered, with nothing or minute quantity
Remnants are qualification.Each liquid volume is：1mL sputums, 2mL fixatives, 2mL liquefiers, 5mLDNAase I enzyme buffer liquids.
Addition Hoechst 33342 dyes 10min after the sputum that liquefies detaches, and 3000rpm centrifuges 3min, and PBS is washed once, and 1/
50 volume PBS hang, and take 1 μ l drops after glass slide, coverslip mounting, liquefaction effect in fluorescence microscopy sputum.
The dyeing observations of Hoechst 33342 show that cell is more complete, have no the filamental compound that DNA is formed with albumen.
Above embodiment be only the preferred embodiment of the present invention is described, not to the scope of the present invention into
Row limits, under the premise of not departing from design spirit of the present invention, technical side of this field ordinary engineering and technical personnel to the present invention
The all variations and modifications that case is made should all be fallen into the protection domain of claims of the present invention determination.
Claims (10)
1. a kind of sputum liquefaction agent, which is characterized in that including fixative, liquefier, bead, Exonucleolytic enzyme buffer liquid；
Contain solvent and polymer in the fixative, the polymer is poly (sodium aspartate) or Poly-L-glutamic acid；The liquefier
In be 7.0~8.5 containing reducing agent and pH value buffer solution, the reducing agent is dithiothreitol (DTT), three (2- carboxyethyls) phosphines
One kind in hydrochloride, dithioerythritol.
2. sputum liquefaction agent according to claim 1, which is characterized in that the solvent in the fixative is volumetric concentration
40~60% ethanol solution, a concentration of 10~1000 μm ol/L of the poly-aspartate in the solvent, poly (sodium aspartate)
Molecular weight be 2000~50000；A concentration of 100~1000 μm ol/L of the Poly-L-glutamic acid in the solvent, molecule
Amount is 5000~1,500,000.
3. sputum liquefaction agent according to claim 1, which is characterized in that contain reducing agent and pH value in the liquefier
For 7.6~8.0 buffer solution, the buffer solution is phosphate or NaHCO3Buffer solution；The reducing agent is selected from 0.05
The dithiothreitol (DTT) of~0.2g/100mL, three (2- carboxyethyls) phosphonium salt hydrochlorates of molar concentration 5-200mmol/L, 0.05~
One kind in the dithioerythritol of 0.2g/100mL.
4. sputum liquefaction agent according to claim 1, which is characterized in that a diameter of 1~5mm of the bead.
5. sputum liquefaction agent according to claim 1, which is characterized in that contain in every milliliter of Exonucleolytic enzyme buffer liquid
The exonuclease of 0~20 unit, the exonuclease are recombinant DNase I.
6. according to Claims 1 to 5 any one of them sputum liquefaction agent, which is characterized in that the fixative 1 including parts by volume
~3 parts, 1~3 part of liquefier, 2~6 parts of bead, 3~8 parts of Exonucleolytic enzyme buffer liquid.
7. a kind of sputum liquefaction agent box, which is characterized in that including claim 1~6 any one of them sputum liquefaction agent,
And aseptic filtration net, the aperture of the net that is sterile filtered is 40~100 μm.
8. sputum liquefaction agent box is in sputum described in claim 1~6 any one of them sputum liquefaction agent or claim 7
Application in liquefaction.
9. the method that application claim 1~6 any one of them sputum liquefaction agent carries out sputum liquefaction, which is characterized in that
Including operation：
1) sputum that volume is 0.8~1.2 times of fixative is transferred in container, fixer and liquefier is added, adds glass
Glass pearl is liquefied 15~30 minutes；
2) be added Exonucleolytic enzyme buffer liquid, observe chamber wall on without azelon silk adherency and liquefier in without agglomerate exist,
Use filter screen filtration.
10. according to the method described in claim 9, it is characterized in that, step 1) liquefaction instrument be vertical rotary mixed instrument,
Vertical rotating speed is 10~50r/min；
After Exonucleolytic enzyme buffer liquid is added in step 2), 25~37 DEG C of 15~30min of placement.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810161127.XA CN108414313A (en) | 2018-02-27 | 2018-02-27 | A kind of sputum liquefaction agent, the kit containing it and its application |
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| CN201810161127.XA CN108414313A (en) | 2018-02-27 | 2018-02-27 | A kind of sputum liquefaction agent, the kit containing it and its application |
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| CN109762809A (en) * | 2019-03-22 | 2019-05-17 | 北京医院 | The extracting method of cfDNA in sputum sample |
| CN111122879A (en) * | 2020-03-26 | 2020-05-08 | 珠海丽珠试剂股份有限公司 | Coating liquid of novel coronavirus recombinant antigen, pretreatment method, application and product |
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