CA2869472C - Sample preparation for flow cytometry - Google Patents
Sample preparation for flow cytometry Download PDFInfo
- Publication number
- CA2869472C CA2869472C CA2869472A CA2869472A CA2869472C CA 2869472 C CA2869472 C CA 2869472C CA 2869472 A CA2869472 A CA 2869472A CA 2869472 A CA2869472 A CA 2869472A CA 2869472 C CA2869472 C CA 2869472C
- Authority
- CA
- Canada
- Prior art keywords
- sample
- background signal
- reducing
- molecule
- nucleic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 238000000684 flow cytometry Methods 0.000 title claims abstract description 41
- 238000002360 preparation method Methods 0.000 title description 12
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 104
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 104
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 104
- 238000000034 method Methods 0.000 claims abstract description 87
- 244000005700 microbiome Species 0.000 claims abstract description 83
- 239000013618 particulate matter Substances 0.000 claims abstract description 35
- 239000011347 resin Substances 0.000 claims abstract description 32
- 229920005989 resin Polymers 0.000 claims abstract description 32
- 239000000523 sample Substances 0.000 claims description 179
- 238000001514 detection method Methods 0.000 claims description 27
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 21
- 239000012466 permeate Substances 0.000 claims description 21
- 230000002829 reductive effect Effects 0.000 claims description 19
- 239000002245 particle Substances 0.000 claims description 16
- 235000013305 food Nutrition 0.000 claims description 15
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 10
- 150000001555 benzenes Chemical class 0.000 claims description 9
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 8
- -1 alkyl amide Chemical class 0.000 claims description 8
- 238000010791 quenching Methods 0.000 claims description 8
- LISFMEBWQUVKPJ-UHFFFAOYSA-N quinolin-2-ol Chemical group C1=CC=C2NC(=O)C=CC2=C1 LISFMEBWQUVKPJ-UHFFFAOYSA-N 0.000 claims description 8
- 229910052717 sulfur Inorganic materials 0.000 claims description 8
- 239000011593 sulfur Substances 0.000 claims description 8
- 239000012472 biological sample Substances 0.000 claims description 6
- 230000007613 environmental effect Effects 0.000 claims description 6
- 230000035699 permeability Effects 0.000 claims description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 5
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical group OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 claims description 5
- 125000000217 alkyl group Chemical group 0.000 claims description 5
- 229910052799 carbon Inorganic materials 0.000 claims description 5
- 125000004432 carbon atom Chemical group C* 0.000 claims description 5
- 229910052739 hydrogen Inorganic materials 0.000 claims description 5
- 239000001257 hydrogen Substances 0.000 claims description 5
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims description 5
- 230000000171 quenching effect Effects 0.000 claims description 5
- 150000003839 salts Chemical class 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 claims description 5
- 239000002537 cosmetic Substances 0.000 claims description 4
- 239000003638 chemical reducing agent Substances 0.000 claims 1
- 241000894006 Bacteria Species 0.000 abstract description 12
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 7
- 238000010186 staining Methods 0.000 abstract description 5
- 210000004027 cell Anatomy 0.000 description 44
- 238000003556 assay Methods 0.000 description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 23
- 239000011159 matrix material Substances 0.000 description 22
- 239000001974 tryptic soy broth Substances 0.000 description 22
- 108010050327 trypticase-soy broth Proteins 0.000 description 22
- 239000000203 mixture Substances 0.000 description 21
- 230000027455 binding Effects 0.000 description 13
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 13
- 244000052769 pathogen Species 0.000 description 12
- 239000000975 dye Substances 0.000 description 11
- 239000000980 acid dye Substances 0.000 description 10
- 239000002028 Biomass Substances 0.000 description 8
- 239000003795 chemical substances by application Substances 0.000 description 6
- 239000007850 fluorescent dye Substances 0.000 description 6
- 125000000623 heterocyclic group Chemical group 0.000 description 6
- 235000013372 meat Nutrition 0.000 description 6
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- 229920001817 Agar Polymers 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- 238000007747 plating Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 4
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- 238000011534 incubation Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 239000013076 target substance Substances 0.000 description 3
- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 description 2
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000003651 drinking water Substances 0.000 description 2
- 235000020188 drinking water Nutrition 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 238000009830 intercalation Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N naphthalene-acid Natural products C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- 125000001624 naphthyl group Chemical group 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 125000004433 nitrogen atom Chemical group N* 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000011533 pre-incubation Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- CHADEQDQBURGHL-UHFFFAOYSA-N (6'-acetyloxy-3-oxospiro[2-benzofuran-1,9'-xanthene]-3'-yl) acetate Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(OC(C)=O)C=C1OC1=CC(OC(=O)C)=CC=C21 CHADEQDQBURGHL-UHFFFAOYSA-N 0.000 description 1
- CVOFKRWYWCSDMA-UHFFFAOYSA-N 2-chloro-n-(2,6-diethylphenyl)-n-(methoxymethyl)acetamide;2,6-dinitro-n,n-dipropyl-4-(trifluoromethyl)aniline Chemical compound CCC1=CC=CC(CC)=C1N(COC)C(=O)CCl.CCCN(CCC)C1=C([N+]([O-])=O)C=C(C(F)(F)F)C=C1[N+]([O-])=O CVOFKRWYWCSDMA-UHFFFAOYSA-N 0.000 description 1
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 1
- 241000223936 Cryptosporidium parvum Species 0.000 description 1
- 241000016605 Cyclospora cayetanensis Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 244000078673 foodborn pathogen Species 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000011090 industrial biotechnology method and process Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 244000005706 microflora Species 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 230000001717 pathogenic effect Effects 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
- 239000004814 polyurethane Substances 0.000 description 1
- 229920002635 polyurethane Polymers 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000009790 rate-determining step (RDS) Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000007320 rich medium Substances 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Classifications
-
- 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
- C12Q1/06—Quantitative determination
-
- 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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Toxicology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Plural Heterocyclic Compounds (AREA)
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201261620823P | 2012-04-05 | 2012-04-05 | |
| US61/620,823 | 2012-04-05 | ||
| PCT/US2013/035291 WO2013152203A1 (en) | 2012-04-05 | 2013-04-04 | Sample preparation for flow cytometry |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2869472A1 CA2869472A1 (en) | 2013-10-10 |
| CA2869472C true CA2869472C (en) | 2017-07-11 |
Family
ID=49301050
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2869472A Active CA2869472C (en) | 2012-04-05 | 2013-04-04 | Sample preparation for flow cytometry |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP2834367A4 (de) |
| JP (1) | JP6401146B2 (de) |
| CN (1) | CN104379760B (de) |
| AU (1) | AU2013243378B2 (de) |
| CA (1) | CA2869472C (de) |
| IN (1) | IN2014DN08957A (de) |
| WO (1) | WO2013152203A1 (de) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11001881B2 (en) | 2006-08-24 | 2021-05-11 | California Institute Of Technology | Methods for detecting analytes |
| US11525156B2 (en) | 2006-07-28 | 2022-12-13 | California Institute Of Technology | Multiplex Q-PCR arrays |
| WO2008014485A2 (en) | 2006-07-28 | 2008-01-31 | California Institute Of Technology | Multiplex q-pcr arrays |
| US11560588B2 (en) | 2006-08-24 | 2023-01-24 | California Institute Of Technology | Multiplex Q-PCR arrays |
| US9933341B2 (en) | 2012-04-05 | 2018-04-03 | Becton, Dickinson And Company | Sample preparation for flow cytometry |
| CA2905651C (en) * | 2013-03-13 | 2019-01-15 | Becton, Dickinson And Company | A method for improving analysis of microorganisms in complex matrices |
| CN104677809B (zh) * | 2013-11-29 | 2018-01-02 | 中国人民解放军第二军医大学 | 隐球菌活力检测试剂盒及检测方法 |
| US9708647B2 (en) | 2015-03-23 | 2017-07-18 | Insilixa, Inc. | Multiplexed analysis of nucleic acid hybridization thermodynamics using integrated arrays |
| US9499861B1 (en) | 2015-09-10 | 2016-11-22 | Insilixa, Inc. | Methods and systems for multiplex quantitative nucleic acid amplification |
| WO2017155858A1 (en) | 2016-03-07 | 2017-09-14 | Insilixa, Inc. | Nucleic acid sequence identification using solid-phase cyclic single base extension |
| CN107643238A (zh) * | 2017-09-19 | 2018-01-30 | 中山大学附属第医院 | 一种定量检测血液循环微粒浓度的方法 |
| ES2967515T3 (es) * | 2018-08-01 | 2024-04-30 | Sartorius Bioanalytical Instr Inc | Métodos, kits y composiciones de tinción para la evaluación de citometría de flujo de partículas del tamaño de un virus no asociadas utilizando múltiples colorantes fluorogénicos |
| JP2022525322A (ja) | 2019-03-14 | 2022-05-12 | インシリクサ， インコーポレイテッド | 時間ゲート蛍光ベースの検出のための方法およびシステム |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5891738A (en) * | 1995-01-16 | 1999-04-06 | Erkki Soini | Biospecific multiparameter assay method |
| US20030013849A1 (en) * | 1999-10-29 | 2003-01-16 | Ward William W. | Renilla reniformis green fluorescent protein |
| RU2172512C1 (ru) * | 2000-02-01 | 2001-08-20 | Закрытое акционерное общество Научно-производственное объединение "ФоМос" | Цветной спектрозональный галогенсеребряный фотографический материал |
| US6323337B1 (en) * | 2000-05-12 | 2001-11-27 | Molecular Probes, Inc. | Quenching oligonucleotides |
| EP2372361B1 (de) * | 2000-11-17 | 2014-08-06 | Biomoda, Inc. | Zusammensetzungen und Verfahren zum Nachweis einer präkanzerogenen Erkrankung in Zell- und Gewebeproben mit 5-, 10-, 15-, 20-Tetrakis (carboxyphenyl) porphin |
| US7205100B2 (en) * | 2002-10-10 | 2007-04-17 | Advanced Analytical Technologies, Inc. | Methods for reducing background fluorescence |
| US8481302B2 (en) * | 2008-11-03 | 2013-07-09 | General Electric Company | Total bacteria monitoring system |
| GB201005939D0 (en) * | 2010-04-09 | 2010-05-26 | Biostatus Ltd | Method of analysing a cell or other biological material containing nucleic acid |
| EP2582838B1 (de) * | 2010-06-21 | 2014-07-16 | Siemens Healthcare Diagnostics Inc. | Löslicher quencher zur hintergrundreduktion bei qpcr-tests |
-
2013
- 2013-04-04 EP EP13773119.6A patent/EP2834367A4/de not_active Withdrawn
- 2013-04-04 AU AU2013243378A patent/AU2013243378B2/en active Active
- 2013-04-04 IN IN8957DEN2014 patent/IN2014DN08957A/en unknown
- 2013-04-04 CA CA2869472A patent/CA2869472C/en active Active
- 2013-04-04 WO PCT/US2013/035291 patent/WO2013152203A1/en active Application Filing
- 2013-04-04 JP JP2015504727A patent/JP6401146B2/ja active Active
- 2013-04-04 CN CN201380029083.0A patent/CN104379760B/zh active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CA2869472A1 (en) | 2013-10-10 |
| IN2014DN08957A (de) | 2015-05-22 |
| WO2013152203A1 (en) | 2013-10-10 |
| JP6401146B2 (ja) | 2018-10-03 |
| EP2834367A1 (de) | 2015-02-11 |
| JP2015514403A (ja) | 2015-05-21 |
| CN104379760B (zh) | 2018-03-16 |
| CN104379760A (zh) | 2015-02-25 |
| AU2013243378B2 (en) | 2019-04-11 |
| EP2834367A4 (de) | 2016-03-23 |
| AU2013243378A1 (en) | 2014-10-30 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request |
Effective date: 20141002 |
|
| EEER | Examination request |
Effective date: 20141002 |