CN104561230A - Sputum specimen homogenization method and preparation of reagent for medical detection of sputum specimen - Google Patents
Sputum specimen homogenization method and preparation of reagent for medical detection of sputum specimen Download PDFInfo
- Publication number
- CN104561230A CN104561230A CN201510031792.3A CN201510031792A CN104561230A CN 104561230 A CN104561230 A CN 104561230A CN 201510031792 A CN201510031792 A CN 201510031792A CN 104561230 A CN104561230 A CN 104561230A
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- China
- Prior art keywords
- sputum
- digestive system
- sputum specimen
- phlegm
- preparation
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- 206010036790 Productive cough Diseases 0.000 title claims abstract description 46
- 208000024794 sputum Diseases 0.000 title claims abstract description 44
- 210000003802 sputum Anatomy 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 14
- 238000001514 detection method Methods 0.000 title claims abstract description 10
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 239000003153 chemical reaction reagent Substances 0.000 title abstract description 3
- 238000000265 homogenisation Methods 0.000 title abstract 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 claims abstract description 6
- 239000002904 solvent Substances 0.000 claims abstract description 6
- 210000002249 digestive system Anatomy 0.000 claims description 42
- 206010062717 Increased upper airway secretion Diseases 0.000 claims description 28
- 208000026435 phlegm Diseases 0.000 claims description 28
- 239000004615 ingredient Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 230000016994 digestive system process Effects 0.000 claims description 2
- 230000029087 digestion Effects 0.000 abstract description 9
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 abstract description 3
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 abstract description 3
- 102000004142 Trypsin Human genes 0.000 abstract 1
- 108090000631 Trypsin Proteins 0.000 abstract 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 abstract 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 abstract 1
- 235000019796 monopotassium phosphate Nutrition 0.000 abstract 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 abstract 1
- 235000011164 potassium chloride Nutrition 0.000 abstract 1
- 239000001103 potassium chloride Substances 0.000 abstract 1
- 239000008223 sterile water Substances 0.000 abstract 1
- 239000012588 trypsin Substances 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 15
- 230000000694 effects Effects 0.000 description 6
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 4
- 238000007400 DNA extraction Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000005336 cracking Methods 0.000 description 3
- 238000009629 microbiological culture Methods 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000006161 blood agar Substances 0.000 description 2
- 235000019219 chocolate Nutrition 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 229910001385 heavy metal Inorganic materials 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 244000005706 microflora Species 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 210000002345 respiratory system Anatomy 0.000 description 2
- 239000003352 sequestering agent Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 229960004418 trolamine Drugs 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000010339 medical test Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a sputum specimen homogenization method and preparation of a reagent for medical detection of a sputum specimen. The invention further provides a novel sputum digestion solution conforming to clinical detection, and the novel sputum digestion solution is prepared from 0.2% (w/v) of N-acetyl-L-cysteine, 0.1% (w/v) of trypsin, 0.4% (w/v) of triethanolamine, 0.2% (w/v) of potassium chloride, 1.5% (w/v) of potassium dihydrogen phosphate, 0.2% (w/v) of dipotassium hydrogen phosphate and a sterile water solvent.
Description
Technical field
The invention provides a kind of novel phlegm Digestive system meeting clinical detection, it can effective homogeneity sputum, protects the bacterium in sputum injury-free simultaneously, is conducive to the further analysis to sputum sample.
Background technology
Generally adopt nature expectoration method and suction sputum method to collect sputum sample clinically, the sputum sample obtained like this has obvious nonuniformity, and the distribution of the compositions such as bacterium is very uneven.Along with the development of medical test and the raising of Good Laboratory control overflow, the pre-treatment of sample is subject to increasing attention.Observe according to routine work, it is general higher that sputum specimen cultivates positive rate, but clinical coincidence rate is unsatisfactory, often occurs detecting actual pathogenic bacterium in sputum and mislead the situation of clinical diagnosis and treatment.
At present, cultivate to clinical sputum specimen bacteria distribution the commodity phlegm Digestive system used both at home and abroad and mainly produce Sputasol (Liquid) SR0233 by OXOID company of Britain, its composition comprises dithiothreitol (DTT), sodium-chlor, Repone K, Sodium phosphate dibasic, potassium primary phosphate etc.In addition, actual clinical detects or uses trypsinase, Chymotrypsin etc. to digest the way of sputum in experiment in addition.
Although had above-mentioned phlegm Digestive system and digestion method for the detection of sputum specimen, clinical practice result has shown, in the homogeneity performance of these Digestive systems and protection sample, the ability of bacterium is still weak.
Summary of the invention
Through large quantity research and comparison, contriver finds that the major cause of the problems referred to above is that the composition of viscous protein in the cracking such as dithiothreitol (DTT), proteolytic enzyme sputum also has infringement to multiple bacterium thalline, and when these material concentrations are lower, homogeneity effect has deficiency.Inventor has devised a kind of novel phlegm Digestive system meeting clinical detection, the viscous protein matter in cracking sputum is carried out with the N-acetyl-L-cysteine of low concentration and trypsinase combination, and be aided with the lower trolamine of sequestrant toxic to eliminate the detrimentally affect of heavy metal to bacterium, good homogeneity effect can be obtained while guarantee is to the low infringement of the bacterium of sputum.
On the one hand, the invention provides a kind of phlegm Digestive system, its composition comprises N-acetyl-L-cysteine.
Further, this phlegm Digestive system is made up of following ingredients:
Sterilized water solvent.
Again further, this phlegm Digestive system is made up of following ingredients:
Sterilized water solvent.
On the other hand, the invention provides the application of phlegm Digestive system of the present invention in the clinical sputum sample detection kit of preparation.
On the other hand, present invention also offers a kind of sputum sample homogeneity method, use phlegm Digestive system process sputum sample provided by the invention.
Embodiment
The preparation of 1 phlegm Digestive system of the present invention and contrast phlegm Digestive system and use
The preparation of phlegm Digestive system of the present invention
Phlegm Digestive system of the present invention is prepared according to following ingredients list:
Sterilized water solvent.
The bottle phlegm Digestive system made being packed as 10ml/ part is kept in 4 DEG C of refrigerators as stock solution.During use, every 10ml stock solution adds sterilized water 90ml and is mixed with working fluid.In sputum, add equivalent working fluid, digest 20 minutes, 10000rpm/min, centrifugal 5min in 37 DEG C of water-baths, taking precipitate is for microbial culture or be used as DNA extraction.
Contrast Digestive system 1: commercially available Sputasol (Liquid) SR0233 (OXOID), using method illustratively.
The trypsinase normal saline solution of contrast Digestive system 2:0.1% (w/v), in sputum, add equivalent Digestive system during use, digest 20 minutes in 37 DEG C of water-baths, 10000rpm/min, centrifugal 5min, taking precipitate is for microbial culture or be used as DNA extraction.
Contrast Digestive system 3:0.1% (w/v) N-acetyl-L-cysteine normal saline solution, in sputum, add equivalent Digestive system during use, digest 20 minutes in 37 DEG C of water-baths, 10000rpm/min, centrifugal 5min, taking precipitate is for microbial culture or be used as DNA extraction.
Contrast Digestive system 4: the phlegm Digestive system in Chinese patent application No. 201110126201.2 embodiments 1, using method is by the record in this application specification sheets.
2 various phlegm Digestive system digestion effects compare
2.1 homogeneity effects
Get 1 part, suction sputum sample, wash 3 times to remove the respiratory tract normal microflora on sputum surface with 40ml stroke-physiological saline solution.According to the digestion of method described in embodiment 1 sputum.Digested sputum be divided into 10 equal portions be inoculated in the dull and stereotyped and chocolate of blood agar, Mai Kangkai dull and stereotyped on, be placed in 35 DEG C of CO
2cultivate in incubator.Distinguish bacterial classification class by colonial morphology and microscopy after 3 days, record the bacterium species number that every portion of sputum is turned out on three kinds of flat boards.Result is as follows:
| 1 kind | 2 kinds | 3 kinds | Be greater than 3 kinds | |
| Phlegm Digestive system of the present invention | 0 | 1 | 7 | 2 |
| Contrast Digestive system 1 | 1 | 3 | 4 | 2 |
| Contrast Digestive system 2 | 2 | 1 | 5 | 2 |
| Contrast Digestive system 3 | 5 | 2 | 3 | 0 |
| Contrast Digestive system 4 | 2 | 4 | 3 | 1 |
Visible in the sputum sample of phlegm Digestive system digestion of the present invention, in each part, bacterium species number is comparatively close, has 70% to be in same interval (3 kinds), significantly better than contrast phlegm Digestive system.
The protected effect of bacterium in 2.2 pairs of samples
100 parts, nature expectoration sample, washs 3 times to remove the respiratory tract normal microflora on sputum surface with 20ml stroke-physiological saline solution.According to the digestion of method described in embodiment 1 sputum.The sputum of digestion is inoculated in the dull and stereotyped and chocolate of blood agar, Mai Kangkai dull and stereotyped on, be placed in 35 DEG C of CO
2cultivate in incubator.Bacterial classification class is distinguished by colonial morphology and microscopy, the bacterium species number that the sputum recording often kind of method process is turned out on three kinds of flat boards after 3 days.Result is as follows:
| 1 kind | 2 kinds | 3 kinds | Be greater than 3 kinds | |
| Phlegm Digestive system of the present invention | 3 | 29 | 51 | 17 |
| Contrast Digestive system 1 | 24 | 24 | 43 | 9 |
| Contrast Digestive system 2 | 30 | 27 | 40 | 3 |
| Contrast Digestive system 3 | 16 | 31 | 45 | 8 |
| Contrast Digestive system 4 | 28 | 19 | 43 | 10 |
Result shows that the bacterial classification class that phlegm Digestive system of the present invention digestion sputum specimen obtains is obviously many compared with contrast Digestive system, demonstrates phlegm Digestive system of the present invention less to the infringement of bacterium in sample.
Above-described embodiment proves, the novel phlegm Digestive system meeting clinical detection of the present invention is by carrying out the viscous protein matter in cracking sputum with the N-acetyl-L-cysteine of low concentration and trypsinase combination, and be aided with the lower trolamine of sequestrant toxic to eliminate the detrimentally affect of heavy metal to bacterium, good homogeneity effect can be obtained while guarantee is to the low infringement of the bacterium in sputum.There is situation in that can react bacterium in sputum more accurately for clinical detection, effectively reduces misdiagnosis rate.
Claims (5)
1. a phlegm Digestive system, its composition comprises N-acetyl-L-cysteine.
2. the phlegm Digestive system of claim 1, is made up of following ingredients:
Sterilized water solvent.
3. the phlegm Digestive system of claim 2, is made up of following ingredients:
Sterilized water solvent.
4. the phlegm Digestive system of claim 1-3 is in the application preparing clinical sputum sample detection kit.
5. a sputum sample homogeneity method, uses the phlegm Digestive system process sputum sample of claim 1-3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510031792.3A CN104561230B (en) | 2015-01-22 | 2015-01-22 | A kind of medical science detects the preparation of sputum sample homogeneity method and reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510031792.3A CN104561230B (en) | 2015-01-22 | 2015-01-22 | A kind of medical science detects the preparation of sputum sample homogeneity method and reagent |
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| Publication Number | Publication Date |
|---|---|
| CN104561230A true CN104561230A (en) | 2015-04-29 |
| CN104561230B CN104561230B (en) | 2015-09-02 |
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| CN201510031792.3A Expired - Fee Related CN104561230B (en) | 2015-01-22 | 2015-01-22 | A kind of medical science detects the preparation of sputum sample homogeneity method and reagent |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105506085A (en) * | 2015-12-28 | 2016-04-20 | 北京澳利文生物科技有限公司 | Treating method for viscous fluid specimen |
| CN106868090A (en) * | 2017-03-28 | 2017-06-20 | 王绪友 | A kind of medical test sputum processing method |
| CN106967778A (en) * | 2017-03-27 | 2017-07-21 | 南京邦博生物技术有限公司 | A kind of quick sputum dissolving preserves liquid |
| CN107400695A (en) * | 2017-03-06 | 2017-11-28 | 李齐 | A kind of sputum microorganism sample homogeneity reagent |
| 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 |
| CN111238893A (en) * | 2020-01-19 | 2020-06-05 | 湖北泰康医疗设备有限公司 | Method for extracting humoral cells for detecting lung cancer |
| WO2022068751A1 (en) * | 2020-09-29 | 2022-04-07 | 郑州安图生物工程股份有限公司 | Method for digesting sputum sample |
| WO2022068746A1 (en) * | 2020-09-29 | 2022-04-07 | 郑州安图生物工程股份有限公司 | Composite sputum digestion liquid |
-
2015
- 2015-01-22 CN CN201510031792.3A patent/CN104561230B/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| 桂勇: "三种细菌培养痰标本预处理方法学评价", 《中国现代医生》 * |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105506085A (en) * | 2015-12-28 | 2016-04-20 | 北京澳利文生物科技有限公司 | Treating method for viscous fluid specimen |
| CN107400695A (en) * | 2017-03-06 | 2017-11-28 | 李齐 | A kind of sputum microorganism sample homogeneity reagent |
| CN107400695B (en) * | 2017-03-06 | 2018-03-06 | 李齐 | A kind of sputum sample homogeneity method and the reagent used |
| CN106967778B (en) * | 2017-03-27 | 2018-04-06 | 南京邦博生物技术有限公司 | A kind of quick sputum dissolving preserves liquid |
| CN106967778A (en) * | 2017-03-27 | 2017-07-21 | 南京邦博生物技术有限公司 | A kind of quick sputum dissolving preserves liquid |
| CN106868090A (en) * | 2017-03-28 | 2017-06-20 | 王绪友 | A kind of medical test sputum processing method |
| CN106868090B (en) * | 2017-03-28 | 2020-09-29 | 王绪友 | Sputum treatment method for medical examination |
| 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 |
| CN111238893A (en) * | 2020-01-19 | 2020-06-05 | 湖北泰康医疗设备有限公司 | Method for extracting humoral cells for detecting lung cancer |
| WO2022068751A1 (en) * | 2020-09-29 | 2022-04-07 | 郑州安图生物工程股份有限公司 | Method for digesting sputum sample |
| WO2022068746A1 (en) * | 2020-09-29 | 2022-04-07 | 郑州安图生物工程股份有限公司 | Composite sputum digestion liquid |
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| CN104561230B (en) | 2015-09-02 |
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