CN107400695A - A kind of sputum microorganism sample homogeneity reagent - Google Patents
A kind of sputum microorganism sample homogeneity reagent Download PDFInfo
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- CN107400695A CN107400695A CN201710128847.1A CN201710128847A CN107400695A CN 107400695 A CN107400695 A CN 107400695A CN 201710128847 A CN201710128847 A CN 201710128847A CN 107400695 A CN107400695 A CN 107400695A
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- Prior art keywords
- sputum
- digestive juice
- phlegm
- reagent
- bacterium
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
Abstract
The invention provides a kind of sputum sample homogeneity method and the reagent used, reagent is by N acetyl L cysteines 0.2% (w/v), trypsase 0.1% (w/v), triethanolamine 0.4% (w/v), potassium chloride 0.2% (w/v), potassium dihydrogen phosphate 1.5% (w/v), phosphoric acid hydrogen dimethylamino 0.2% (w/v), sterilized water solvent composition.
Description
Technical field
The present invention relates to for a kind of clinical sputum sample sputum sample homogeneity method handled and the reagent used, its
Can effective homogeneity sputum, while the bacterium for protecting sputum to include including Legionella is injury-free, is advantageous to sputum sample
Further analysis.
Background technology
The sputum sample for collecting sputum sample acquisition using nature expectoration method and suction sputum method in breathing problem inspection has
The distribution of the compositions such as obvious anisotropism, bacterium is very uneven, generally requires sample homogeneity using phlegm digestive juice.With
The raising of accuracy and quality control of the experiment requirement is checked, the pre-treatment of sample is more and more paid attention to, according to daily work
Observe, sputum specimen cultivation results and still undesirable with actual clinical coincidence rate, often occur detecting in sputum
Actual pathogenic bacteria and mislead the situation of clinical diagnosis and treatment.
At present, both at home and abroad to commodity phlegm digestive juice used in clinical sputum specimen bacteria distribution culture mainly by Britain
OXOID companies produce Sputasol (Liquid) SR0233, and its composition includes dithiothreitol (DTT), sodium chloride, potassium chloride, phosphoric acid hydrogen
Disodium, potassium dihydrogen phosphate etc..In addition, also have in actual clinical detection or experiment using the digestion such as trypsase, chymotrypsin
The way of sputum.Chinese patent application 201110126201.2 provides a kind of phlegm digestive juice suitable for Legionella, its
Sodium salt is replaced with sylvite on the basis of Sputasol (Liquid) SR0233, and adds EDTA, to avoid sodium ion and heavy metal
Ion pair Legionella and the toxicity of other bacterium simultaneously strengthen homogeneity effect.Chinese patent application 201510031792.3 then uses N-
Acetyl group-L cysteines, triethanolamine, trypsase digest sputum, preferably protect the microorganism in sputum.
Although having there is the detection that above-mentioned phlegm digestive juice and digestion method are used for sputum specimen, clinical practice result shows,
Bacterium in the homogeneity performances of these digestive juices and protection sample (it is particularly some in external environment compared with the bacterium of easy in inactivation, such as army
Group bacterium) ability be still weak.And dithiothreitol (DTT) ingredient stability is poor, the reagent holding time is shorter.
The content of the invention
Inventor through comparison and screening test devises a kind of sputum sample homogeneity method and the reagent used, with compared with
The TCEP hydrochlorides (three (2- acyls ethyl) phosphonium salt hydrochlorates) and trypsase of low concentration are combined to crack the viscous protein in sputum
Matter, and it is aided with biosurfactant/chelating agent rhamnolipid to eliminate harmful effect of the metal ion to bacterium and strengthen digestion
Effect, it can ensure to obtaining good homogeneity effect while the low infringement of bacterium including Legionella.
On the one hand, the invention provides a kind of phlegm digestive juice, its composition to include TECP hydrochlorides.
Further, the phlegm digestive juice is made up of following ingredients：
Yet further, the phlegm digestive juice is made up of following ingredients：
On the other hand, the invention provides the phlegm digestive juice of the present invention to prepare answering for clinical sputum sample detection kit
With.
On the other hand, present invention also offers a kind of sputum sample homogeneity method, digested using phlegm provided by the invention
Liquid handles sputum sample.
Embodiment
The phlegm digestive juice of the present invention of embodiment 1 and the preparation and use for compareing phlegm digestive juice
The preparation of the phlegm digestive juice of the present invention
The phlegm digestive juice of the present invention is prepared according to following ingredients list：
Various reagents are that domestic analysis is pure, from Chinese medicines group, brag holy biology etc.
The phlegm digestive juice made is packed as into 15ml/ parts to be stored in 4 DEG C of refrigerators.In use, every part of storage plus sterilized water
50ml is configured to working solution.Add approximately equal amount working solution into sputum, digested 15 minutes, 10000rpm/min in 37 DEG C of water-baths,
Centrifuge 5min, you can taking precipitate extracts use for Bacteria Culture or as DNA.
Embodiment 2 is compared with existing phlegm digestive juice digestion effect
Commercially available Sputasol (Liquid) SR0233 (OXOID), application method is illustratively.
Digestive juice 1：Phlegm digestive juice in No. 201110126201.2 embodiments 1 of Chinese patent application, application method is pressed should
Record in application specification.
Digestive juice 2：Phlegm digestive juice in No. 201510031792.3 embodiments 1 of Chinese patent application, application method is pressed should
Record in application specification.
2.1 homogeneity effects
Take 1 part of suction sputum sample (volume about 40ml), be divided into 4 equal portions, respectively with 200ml sterile salines wash 3 times with
Remove the respiratory tract normal flora on sputum surface.According to method described in embodiment 1 and Sputasol (Liquid) SR0233
(OXOID), Chinese patent application 201110126201.2, the method for No. 201510031792.3 records of Chinese patent application disappear
Resolving sputum liquid.The sputum of every kind of digestion be divided into 9 equal portions be inoculated on blood plate, Mai Kangkai flat boards and chocolate flat board it is (every kind of flat
3 parts of plate), it is placed in 35 DEG C of CO2Cultivated in incubator.Strain class is distinguished by colonial morphology and microscopy after 3 days, records every portion of sputum
The bacterium species number turned out on three kinds of flat boards.As a result it is as follows：
It can be seen that in the sputum sample that the phlegm digestive juice through the present invention digests, and bacterium species number is closer in each part, greatly
It is partially in 3 kinds of section, and detects strain quantity at most (29 kinds), hence it is evident that is better than other several phlegm digestive juices.
The protecting effect of bacterium in 2.2 pairs of samples
80 parts of the natural expectoration sample (every part of about 5ml) taken in division of respiratory disease procedure, it is equally divided into 4 groups and is used for 4 kinds
Digestive juice is tested, and every part is washed 3 times to remove the respiratory tract normal flora on sputum surface with 20ml sterile salines respectively.Press
According to method described in embodiment 1 and Sputasol (Liquid) SR0233 (OXOID), Chinese patent application
201110126201.2 number, the method digestion sputum of the record of Chinese patent application 201510031792.3.By the sputum of digestion
It is divided into 3 equal portions to be inoculated on blood plate, Mai Kangkai flat boards and chocolate flat board, is placed in 35 DEG C of CO2Cultivated in incubator.Lead to after 3 days
Crossing colonial morphology, to distinguish strain class, (the similar strain in identical sample is included in once, the similar bacterium in different samples with microscopy
Kind is included in respectively), record the bacterium species number that the sputum of every kind of method processing is turned out on three kinds of flat boards.As a result it is as follows：
As a result show that the strain class that the phlegm digestive juice digestion sputum specimen of the present invention obtains is substantially more compared with control digestive juice (near
200 kinds), it was demonstrated that infringement of the phlegm digestive juice of the present invention to bacterium in sample is smaller.
The protecting effect of Legionella in 2.3 pairs of samples
10 parts of the natural expectoration sample with Legionella is determined with PCR joint-detections through cultivating, described in embodiment 1
Method and Sputasol (Liquid) SR0233 (OXOID), Chinese patent application 201110126201.2, Chinese patent Shen
Please No. 201510031792.3 method digestion sputums recorded.The sputum of digestion is inoculated in Chinese patent application
201010192273.2 number legion's bacterium culture medium in.Through culture, there are 8 parts of detections in the sample of phlegm digestive juice of the invention processing
To Legionella, Sputasol (Liquid) SR0233 (OXOID), 201110126201.2,201010192273.2 only have respectively
3rd, 5,3 parts detect Legionella.
As a result show still to obtain good for the Legionella weaker to extraneous environmental resistance, phlegm digestive juice of the invention
Good Detection results, no less than the special phlegm digestive juice of Legionella in Chinese patent application 201110126201.2, better than
General phlegm digestive juice.
Above-described embodiment proves that sputum sample homogeneity method of the invention and the reagent used pass through with low concentration
TCEP hydrochlorides (three (2- acyls ethyl) phosphonium salt hydrochlorates) and trypsase are combined to crack the viscous protein matter in sputum, and are aided with
Biosurfactant/chelating agent rhamnolipid eliminates harmful effect of the metal ion to bacterium and strengthens digestion effect, can be with
Ensureing to obtaining good homogeneity effect while the low infringement of bacterium including Legionella.Can be more accurate for clinical detection
The presence situation of bacterium, effectively reduces misdiagnosis rate in true reaction sputum, especially can be to improve the Detection results to Legionella.
Claims (4)
1. a kind of phlegm digestive juice, its composition includes TECP hydrochlorides.
2. the phlegm digestive juice of claim 1, is made up of following ingredients：
3. the phlegm digestive juice of claim 1 or 2 is preparing the application of clinical sputum sample detection kit.
4. a kind of sputum sample homogeneity method, the phlegm digestive juice processing sputum sample of usage right requirement 1 or 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710128847.1A CN107400695B (en) | 2017-03-06 | 2017-03-06 | A kind of sputum sample homogeneity method and the reagent used |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710128847.1A CN107400695B (en) | 2017-03-06 | 2017-03-06 | A kind of sputum sample homogeneity method and the reagent used |
Publications (2)
| Publication Number | Publication Date |
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| CN107400695A true CN107400695A (en) | 2017-11-28 |
| CN107400695B CN107400695B (en) | 2018-03-06 |
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| CN201710128847.1A Expired - Fee Related CN107400695B (en) | 2017-03-06 | 2017-03-06 | A kind of sputum sample homogeneity method and the reagent used |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109679946A (en) * | 2019-01-04 | 2019-04-26 | 宁波艾捷康宁生物科技有限公司 | A kind of blood disease RAN protective agent and heparin tube |
| CN110964842A (en) * | 2019-12-31 | 2020-04-07 | 广州金域医学检验集团股份有限公司 | Sample processing reagent and kit for detecting mycobacterium tuberculosis nucleic acid and nucleic acid amplification method of mycobacterium tuberculosis |
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| WO2011144304A1 (en) * | 2010-05-17 | 2011-11-24 | Curetis Ag | Universally applicable lysis buffer and processing methods for the lysis of bodily samples |
| CN104531659A (en) * | 2014-12-16 | 2015-04-22 | 四川新健康成生物股份有限公司 | Trypsin digestive juice, preparation method and ready-to-use phlegm digesting device |
| CN104561230A (en) * | 2015-01-22 | 2015-04-29 | 四川金域医学检验中心有限公司 | Sputum specimen homogenization method and preparation of reagent for medical detection of sputum specimen |
| CN105274185A (en) * | 2015-11-16 | 2016-01-27 | 南京邦博生物技术有限公司 | Sputum treating liquid for detecting Mycobacterium tuberculosis |
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2017
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Patent Citations (4)
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|---|---|---|---|---|
| WO2011144304A1 (en) * | 2010-05-17 | 2011-11-24 | Curetis Ag | Universally applicable lysis buffer and processing methods for the lysis of bodily samples |
| CN104531659A (en) * | 2014-12-16 | 2015-04-22 | 四川新健康成生物股份有限公司 | Trypsin digestive juice, preparation method and ready-to-use phlegm digesting device |
| CN104561230A (en) * | 2015-01-22 | 2015-04-29 | 四川金域医学检验中心有限公司 | Sputum specimen homogenization method and preparation of reagent for medical detection of sputum specimen |
| CN105274185A (en) * | 2015-11-16 | 2016-01-27 | 南京邦博生物技术有限公司 | Sputum treating liquid for detecting Mycobacterium tuberculosis |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109679946A (en) * | 2019-01-04 | 2019-04-26 | 宁波艾捷康宁生物科技有限公司 | A kind of blood disease RAN protective agent and heparin tube |
| CN109679946B (en) * | 2019-01-04 | 2020-11-13 | 宁波艾捷康宁生物科技有限公司 | Blood virus RNA protective agent and blood collection tube |
| CN110964842A (en) * | 2019-12-31 | 2020-04-07 | 广州金域医学检验集团股份有限公司 | Sample processing reagent and kit for detecting mycobacterium tuberculosis nucleic acid and nucleic acid amplification method of mycobacterium tuberculosis |
| CN110964842B (en) * | 2019-12-31 | 2023-11-03 | 广州金域医学检验集团股份有限公司 | Sample processing reagent for detecting mycobacterium tuberculosis nucleic acid, kit and mycobacterium tuberculosis nucleic acid amplification method |
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