EP3942034A1 - Billes de verre fonctionnalisees, leur utilisation pour capter des micro-organismes et les dispositifs correspondants - Google Patents
Billes de verre fonctionnalisees, leur utilisation pour capter des micro-organismes et les dispositifs correspondantsInfo
- Publication number
- EP3942034A1 EP3942034A1 EP20710190.8A EP20710190A EP3942034A1 EP 3942034 A1 EP3942034 A1 EP 3942034A1 EP 20710190 A EP20710190 A EP 20710190A EP 3942034 A1 EP3942034 A1 EP 3942034A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- microorganisms
- lysine
- polylysine
- glass beads
- approximately
- 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.)
- Pending
Links
- 244000005700 microbiome Species 0.000 title claims abstract description 218
- 239000011324 bead Substances 0.000 title claims abstract description 171
- 239000011521 glass Substances 0.000 title claims abstract description 166
- 108010039918 Polylysine Proteins 0.000 claims description 96
- 229920000656 polylysine Polymers 0.000 claims description 96
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 82
- 239000004472 Lysine Substances 0.000 claims description 79
- 238000010828 elution Methods 0.000 claims description 79
- 235000018977 lysine Nutrition 0.000 claims description 72
- 238000000034 method Methods 0.000 claims description 67
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 63
- 239000000243 solution Substances 0.000 claims description 58
- 239000000523 sample Substances 0.000 claims description 57
- 229920000729 poly(L-lysine) polymer Polymers 0.000 claims description 43
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 40
- 239000007788 liquid Substances 0.000 claims description 39
- 230000008030 elimination Effects 0.000 claims description 30
- 238000003379 elimination reaction Methods 0.000 claims description 30
- 238000003745 diagnosis Methods 0.000 claims description 26
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 24
- 230000008569 process Effects 0.000 claims description 16
- 239000000203 mixture Substances 0.000 claims description 15
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine Chemical compound NCCCC[C@@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 claims description 14
- 235000019766 L-Lysine Nutrition 0.000 claims description 12
- 239000000126 substance Substances 0.000 claims description 12
- 235000013305 food Nutrition 0.000 claims description 11
- 230000009467 reduction Effects 0.000 claims description 11
- 241000607142 Salmonella Species 0.000 claims description 10
- 230000002255 enzymatic effect Effects 0.000 claims description 10
- 241000894006 Bacteria Species 0.000 claims description 9
- -1 urine Substances 0.000 claims description 9
- 241000233866 Fungi Species 0.000 claims description 8
- 230000003993 interaction Effects 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 7
- 239000002537 cosmetic Substances 0.000 claims description 7
- 238000011084 recovery Methods 0.000 claims description 7
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 claims description 6
- 241000191940 Staphylococcus Species 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 6
- HUAUNKAZQWMVFY-UHFFFAOYSA-M sodium;oxocalcium;hydroxide Chemical compound [OH-].[Na+].[Ca]=O HUAUNKAZQWMVFY-UHFFFAOYSA-M 0.000 claims description 6
- 125000001176 L-lysyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C([H])([H])C([H])([H])C([H])([H])C(N([H])[H])([H])[H] 0.000 claims description 5
- 239000012472 biological sample Substances 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 235000013336 milk Nutrition 0.000 claims description 5
- 239000008267 milk Substances 0.000 claims description 5
- 210000004080 milk Anatomy 0.000 claims description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 4
- 235000015203 fruit juice Nutrition 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 241000228143 Penicillium Species 0.000 claims description 3
- 241000235648 Pichia Species 0.000 claims description 3
- 241000233639 Pythium Species 0.000 claims description 3
- 244000269722 Thea sinensis Species 0.000 claims description 3
- 235000013334 alcoholic beverage Nutrition 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 3
- 239000006071 cream Substances 0.000 claims description 3
- 235000013365 dairy product Nutrition 0.000 claims description 3
- 239000002781 deodorant agent Substances 0.000 claims description 3
- 235000013601 eggs Nutrition 0.000 claims description 3
- 239000003889 eye drop Substances 0.000 claims description 3
- 229940012356 eye drops Drugs 0.000 claims description 3
- 239000000499 gel Substances 0.000 claims description 3
- 210000002751 lymph Anatomy 0.000 claims description 3
- 235000013372 meat Nutrition 0.000 claims description 3
- 210000004400 mucous membrane Anatomy 0.000 claims description 3
- 235000015504 ready meals Nutrition 0.000 claims description 3
- 230000028327 secretion Effects 0.000 claims description 3
- 239000002453 shampoo Substances 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 210000001179 synovial fluid Anatomy 0.000 claims description 3
- 239000006188 syrup Substances 0.000 claims description 3
- 235000020357 syrup Nutrition 0.000 claims description 3
- 210000001138 tear Anatomy 0.000 claims description 3
- 229960005486 vaccine Drugs 0.000 claims description 3
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 claims description 2
- 241000235389 Absidia Species 0.000 claims description 2
- 241000589220 Acetobacter Species 0.000 claims description 2
- 241000590020 Achromobacter Species 0.000 claims description 2
- 241000726119 Acidovorax Species 0.000 claims description 2
- 241000589291 Acinetobacter Species 0.000 claims description 2
- 241000186046 Actinomyces Species 0.000 claims description 2
- 241000193798 Aerococcus Species 0.000 claims description 2
- 241000607534 Aeromonas Species 0.000 claims description 2
- 241000588986 Alcaligenes Species 0.000 claims description 2
- 241001147780 Alicyclobacillus Species 0.000 claims description 2
- 241000223600 Alternaria Species 0.000 claims description 2
- 241000228212 Aspergillus Species 0.000 claims description 2
- 241000223651 Aureobasidium Species 0.000 claims description 2
- 241000588807 Bordetella Species 0.000 claims description 2
- 241001465180 Botrytis Species 0.000 claims description 2
- 241000157902 Brachybacterium Species 0.000 claims description 2
- 241000722885 Brettanomyces Species 0.000 claims description 2
- 241000555281 Brevibacillus Species 0.000 claims description 2
- 241000186146 Brevibacterium Species 0.000 claims description 2
- 241000131407 Brevundimonas Species 0.000 claims description 2
- 241001453380 Burkholderia Species 0.000 claims description 2
- 241001622847 Buttiauxella Species 0.000 claims description 2
- 241000228337 Byssochlamys Species 0.000 claims description 2
- 241000589876 Campylobacter Species 0.000 claims description 2
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 2
- 241000206594 Carnobacterium Species 0.000 claims description 2
- 241000186321 Cellulomonas Species 0.000 claims description 2
- 241000221955 Chaetomium Species 0.000 claims description 2
- 241000588923 Citrobacter Species 0.000 claims description 2
- 241000222290 Cladosporium Species 0.000 claims description 2
- 241000186650 Clavibacter Species 0.000 claims description 2
- 241000193403 Clostridium Species 0.000 claims description 2
- 241000222199 Colletotrichum Species 0.000 claims description 2
- 241000186216 Corynebacterium Species 0.000 claims description 2
- 241000235035 Debaryomyces Species 0.000 claims description 2
- 241000228138 Emericella Species 0.000 claims description 2
- 241001492222 Epicoccum Species 0.000 claims description 2
- 241001136487 Eurotium Species 0.000 claims description 2
- 241000589565 Flavobacterium Species 0.000 claims description 2
- 241000223218 Fusarium Species 0.000 claims description 2
- 241001123633 Galactomyces Species 0.000 claims description 2
- 241000626621 Geobacillus Species 0.000 claims description 2
- 241000159512 Geotrichum Species 0.000 claims description 2
- 241000193004 Halobacillus Species 0.000 claims description 2
- 241001149669 Hanseniaspora Species 0.000 claims description 2
- 241000588748 Klebsiella Species 0.000 claims description 2
- 241000579722 Kocuria Species 0.000 claims description 2
- 241000186660 Lactobacillus Species 0.000 claims description 2
- 241000194036 Lactococcus Species 0.000 claims description 2
- 241001647840 Leclercia Species 0.000 claims description 2
- 241000033241 Lelliottia Species 0.000 claims description 2
- 241000192132 Leuconostoc Species 0.000 claims description 2
- 241000568397 Lysinibacillus Species 0.000 claims description 2
- 241000973040 Macrococcus Species 0.000 claims description 2
- 241001467578 Microbacterium Species 0.000 claims description 2
- 241000192041 Micrococcus Species 0.000 claims description 2
- 241000186359 Mycobacterium Species 0.000 claims description 2
- 241000579725 Nesterenkonia Species 0.000 claims description 2
- 241001072230 Oceanobacillus Species 0.000 claims description 2
- 241000588843 Ochrobactrum Species 0.000 claims description 2
- 241000179039 Paenibacillus Species 0.000 claims description 2
- 241000520272 Pantoea Species 0.000 claims description 2
- 241001057811 Paracoccus <mealybug> Species 0.000 claims description 2
- 241000192001 Pediococcus Species 0.000 claims description 2
- 241001503951 Phoma Species 0.000 claims description 2
- 241000233614 Phytophthora Species 0.000 claims description 2
- 241000186429 Propionibacterium Species 0.000 claims description 2
- 241000588769 Proteus <enterobacteria> Species 0.000 claims description 2
- 241000589516 Pseudomonas Species 0.000 claims description 2
- 241000232299 Ralstonia Species 0.000 claims description 2
- 241000589180 Rhizobium Species 0.000 claims description 2
- 241001361634 Rhizoctonia Species 0.000 claims description 2
- 241000235527 Rhizopus Species 0.000 claims description 2
- 241000223252 Rhodotorula Species 0.000 claims description 2
- 241000220317 Rosa Species 0.000 claims description 2
- 241000235070 Saccharomyces Species 0.000 claims description 2
- 241000235003 Saccharomycopsis Species 0.000 claims description 2
- 241000235346 Schizosaccharomyces Species 0.000 claims description 2
- 241000607720 Serratia Species 0.000 claims description 2
- 241000863430 Shewanella Species 0.000 claims description 2
- 241000736131 Sphingomonas Species 0.000 claims description 2
- 241000204117 Sporolactobacillus Species 0.000 claims description 2
- 241000186547 Sporosarcina Species 0.000 claims description 2
- 241000122971 Stenotrophomonas Species 0.000 claims description 2
- 241000194017 Streptococcus Species 0.000 claims description 2
- 241000187747 Streptomyces Species 0.000 claims description 2
- 241001137870 Thermoanaerobacterium Species 0.000 claims description 2
- 241000266300 Ulocladium Species 0.000 claims description 2
- 241001478283 Variovorax Species 0.000 claims description 2
- 241000082085 Verticillium <Phyllachorales> Species 0.000 claims description 2
- 241001659629 Virgibacillus Species 0.000 claims description 2
- 241001339206 Wallemia Species 0.000 claims description 2
- 229930194204 Wallemia Natural products 0.000 claims description 2
- 241000370151 Wickerhamomyces Species 0.000 claims description 2
- 241001523965 Xylaria Species 0.000 claims description 2
- 241000235017 Zygosaccharomyces Species 0.000 claims description 2
- 238000004140 cleaning Methods 0.000 claims description 2
- 229940039696 lactobacillus Drugs 0.000 claims description 2
- 230000037072 sun protection Effects 0.000 claims description 2
- 210000002700 urine Anatomy 0.000 claims description 2
- 241000186000 Bifidobacterium Species 0.000 claims 1
- 241001337994 Cryptococcus <scale insect> Species 0.000 claims 1
- 241001528480 Cupriavidus Species 0.000 claims 1
- 241000203813 Curtobacterium Species 0.000 claims 1
- 241000966210 Elizabethkingia Species 0.000 claims 1
- 241000122379 Enteractinococcus Species 0.000 claims 1
- 241000588914 Enterobacter Species 0.000 claims 1
- 241000194033 Enterococcus Species 0.000 claims 1
- 241000588722 Escherichia Species 0.000 claims 1
- 241000658549 Mycocladus Species 0.000 claims 1
- 241001507755 Neosartorya Species 0.000 claims 1
- 241000189150 Nigrospora Species 0.000 claims 1
- 241001236817 Paecilomyces <Clavicipitaceae> Species 0.000 claims 1
- 241000893212 Pestalotia Species 0.000 claims 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 50
- 239000011780 sodium chloride Substances 0.000 description 25
- 239000000725 suspension Substances 0.000 description 23
- 238000001035 drying Methods 0.000 description 19
- 230000007480 spreading Effects 0.000 description 15
- 238000003892 spreading Methods 0.000 description 15
- 238000010790 dilution Methods 0.000 description 14
- 239000012895 dilution Substances 0.000 description 14
- 229920001817 Agar Polymers 0.000 description 13
- 239000008272 agar Substances 0.000 description 13
- 239000000047 product Substances 0.000 description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- 239000012528 membrane Substances 0.000 description 11
- 230000001332 colony forming effect Effects 0.000 description 10
- 238000004163 cytometry Methods 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 239000012466 permeate Substances 0.000 description 10
- 102000004142 Trypsin Human genes 0.000 description 9
- 108090000631 Trypsin Proteins 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 9
- 150000002669 lysines Chemical class 0.000 description 9
- 210000004379 membrane Anatomy 0.000 description 9
- 238000005374 membrane filtration Methods 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 239000012588 trypsin Substances 0.000 description 9
- 235000014469 Bacillus subtilis Nutrition 0.000 description 8
- 238000000684 flow cytometry Methods 0.000 description 8
- 238000007306 functionalization reaction Methods 0.000 description 8
- 230000003068 static effect Effects 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 230000012010 growth Effects 0.000 description 7
- 241000223221 Fusarium oxysporum Species 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 241000235526 Mucor racemosus Species 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000011156 evaluation Methods 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- 229920000642 polymer Polymers 0.000 description 6
- 238000003752 polymerase chain reaction Methods 0.000 description 6
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 5
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 5
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 239000011575 calcium Substances 0.000 description 5
- 229910052791 calcium Inorganic materials 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 5
- 238000012543 microbiological analysis Methods 0.000 description 5
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 5
- 238000001179 sorption measurement Methods 0.000 description 5
- 230000001960 triggered effect Effects 0.000 description 5
- 244000063299 Bacillus subtilis Species 0.000 description 4
- 241000222173 Candida parapsilosis Species 0.000 description 4
- 240000001817 Cereus hexagonus Species 0.000 description 4
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 4
- 238000010240 RT-PCR analysis Methods 0.000 description 4
- 241000607715 Serratia marcescens Species 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 241000191967 Staphylococcus aureus Species 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 239000000470 constituent Substances 0.000 description 4
- 239000011777 magnesium Substances 0.000 description 4
- 229910052749 magnesium Inorganic materials 0.000 description 4
- 230000002906 microbiologic effect Effects 0.000 description 4
- 230000002035 prolonged effect Effects 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 241000025053 Escherichia coli DSM 30083 = JCM 1649 = ATCC 11775 Species 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 238000000265 homogenisation Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 230000035790 physiological processes and functions Effects 0.000 description 3
- 229920001992 poloxamer 407 Polymers 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 2
- 241000194103 Bacillus pumilus Species 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 241000588697 Enterobacter cloacae Species 0.000 description 2
- 241000194031 Enterococcus faecium Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000588770 Proteus mirabilis Species 0.000 description 2
- 241000266403 Salmonella enterica subsp. enterica serovar Choleraesuis Species 0.000 description 2
- 241001354013 Salmonella enterica subsp. enterica serovar Enteritidis Species 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 108010076089 accutase Proteins 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 235000015197 apple juice Nutrition 0.000 description 2
- 235000013361 beverage Nutrition 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Natural products OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- JKWMSGQKBLHBQQ-UHFFFAOYSA-N diboron trioxide Chemical compound O=BOB=O JKWMSGQKBLHBQQ-UHFFFAOYSA-N 0.000 description 2
- 239000003651 drinking water Substances 0.000 description 2
- 235000020188 drinking water Nutrition 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000011901 isothermal amplification Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 235000015205 orange juice Nutrition 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000004611 spectroscopical analysis Methods 0.000 description 2
- 238000009423 ventilation Methods 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- KZEVSDGEBAJOTK-UHFFFAOYSA-N 1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-2-[5-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]-1,3,4-oxadiazol-2-yl]ethanone Chemical compound N1N=NC=2CN(CCC=21)C(CC=1OC(=NN=1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)=O KZEVSDGEBAJOTK-UHFFFAOYSA-N 0.000 description 1
- SXAMGRAIZSSWIH-UHFFFAOYSA-N 2-[3-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,2,4-oxadiazol-5-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NOC(=N1)CC(=O)N1CC2=C(CC1)NN=N2 SXAMGRAIZSSWIH-UHFFFAOYSA-N 0.000 description 1
- YJLUBHOZZTYQIP-UHFFFAOYSA-N 2-[5-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,3,4-oxadiazol-2-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NN=C(O1)CC(=O)N1CC2=C(CC1)NN=N2 YJLUBHOZZTYQIP-UHFFFAOYSA-N 0.000 description 1
- PXRKCOCTEMYUEG-UHFFFAOYSA-N 5-aminoisoindole-1,3-dione Chemical compound NC1=CC=C2C(=O)NC(=O)C2=C1 PXRKCOCTEMYUEG-UHFFFAOYSA-N 0.000 description 1
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 1
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 1
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 1
- 241000589944 Aquaspirillum Species 0.000 description 1
- 241000308413 Asaia Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000131482 Bifidobacterium sp. Species 0.000 description 1
- 208000035985 Body Odor Diseases 0.000 description 1
- 241001527609 Cryptococcus Species 0.000 description 1
- 241000896533 Gliocladium Species 0.000 description 1
- 241000223198 Humicola Species 0.000 description 1
- 241000376403 Hyphopichia Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241000235649 Kluyveromyces Species 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 241001352082 Lichtheimia Species 0.000 description 1
- 241001508815 Lodderomyces Species 0.000 description 1
- 241000311506 Meyerozyma Species 0.000 description 1
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 description 1
- 229920000463 Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) Polymers 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 241000221662 Sclerotinia Species 0.000 description 1
- 241000122799 Scopulariopsis Species 0.000 description 1
- 241001599571 Serpula <basidiomycete> Species 0.000 description 1
- 206010040904 Skin odour abnormal Diseases 0.000 description 1
- 241000371621 Stemphylium Species 0.000 description 1
- 102220606724 Syndecan-1_L27E_mutation Human genes 0.000 description 1
- 241000228341 Talaromyces Species 0.000 description 1
- 241000865903 Thielaviopsis Species 0.000 description 1
- 241000235006 Torulaspora Species 0.000 description 1
- 241000223259 Trichoderma Species 0.000 description 1
- 241000223230 Trichosporon Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 210000004666 bacterial spore Anatomy 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 229920001400 block copolymer Polymers 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 150000001719 carbohydrate derivatives Chemical class 0.000 description 1
- 229920006317 cationic polymer Polymers 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229920001903 high density polyethylene Polymers 0.000 description 1
- 239000004700 high-density polyethylene Substances 0.000 description 1
- 238000003312 immunocapture Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000002241 neurite Anatomy 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 229940044476 poloxamer 407 Drugs 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 210000002243 primary neuron Anatomy 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000005361 soda-lime glass Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000000475 sunscreen effect Effects 0.000 description 1
- 239000000516 sunscreening agent Substances 0.000 description 1
- 238000004381 surface treatment Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- UEUXEKPTXMALOB-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEUXEKPTXMALOB-UHFFFAOYSA-J 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000009736 wetting Methods 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
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/42—Preservation of non-alcoholic beverages
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/70—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter
- A23L2/80—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter by adsorption
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/358—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3598—Apparatus for preserving using solids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/27—Removal of unwanted matter, e.g. deodorisation or detoxification by chemical treatment, by adsorption or by absorption
- A23L5/273—Removal of unwanted matter, e.g. deodorisation or detoxification by chemical treatment, by adsorption or by absorption using adsorption or absorption agents, resins, synthetic polymers, or ion exchangers
-
- G—PHYSICS
- 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
- G01N1/40—Concentrating samples
- G01N1/405—Concentrating samples by adsorption or absorption
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Definitions
- Functionalized glass beads their use to capture microorganisms and the corresponding devices.
- the present invention relates to functionalized glass beads, their preparation process and their use in the capture of microorganisms for the implementation of a process for the elimination of microorganisms or for diagnosis, as well as devices comprising these.
- functionalized glass beads allowing the implementation of these different processes.
- microorganisms The capture of microorganisms is of fundamental interest in many types of industry, such as the pharmaceutical, cosmetic or food industries. It can in particular be used for applications which fall into two main areas:
- the major drawback of heat treatment stems from the fact that the temperature is liable to cause irreversible changes in the product by acting directly on its constituents. Also, at the microbiological level, the heat treatment corresponds to a reduction in the load of microorganisms and it is, for example, capable of reactivating bacterial spores. Regarding membrane filtration, membrane clogging is the main problem that can be encountered. The latter may be due to the concentration of microorganisms in the product as well as to the nature of the product and in particular to the solid particles present within it.
- Microbiological analysis requires the use of precise techniques for which the time to obtain the shortest possible result is sought. Indeed, the faster the analysis results are obtained, the more it is possible to initiate corrective actions in the event of unsatisfactory or unacceptable results.
- it is necessary to predict and diagnose the risk of infection the more rapid and precise the diagnosis, the more effective the care of patients and the minimized risk of transmission.
- a key step for most of these methods for detecting / quantifying microorganisms in liquid or liquid samples is their ability to exceed the detection limit of the evaluation technique used. This is particularly difficult for samples with low concentrations of the microorganisms of interest. An enrichment or concentration step often appears necessary for this type of sample.
- the pre-enrichment and / or enrichment phase requires the use of culture media, selective or not, which aim to promote the growth of target microorganisms in biological or environmental samples, while limiting the growth of flora non-target.
- the target population which is often present at low levels compared to the additional flora present in foods, is amplified.
- Another possibility is to go through a step of concentrating the microorganisms from the liquid or liquid sample.
- One of the most widely used techniques for the concentration of microorganisms from samples is the use of one or more membrane filters of varying porosity through which the liquid medium is filtered. The microorganisms in the sample are stopped by the membrane and therefore concentrated. Such a technique is generally used for the microbiological analysis of process water, drinking water, or drinking water.
- microorganisms can be concentrated and separated from the constituent elements of the sample.
- immunocapture or immunoconcentration methods are widely used in many applications. They very often use supports functionalized with antibodies and make it possible to specifically capture or not the microorganisms contained in the sample. These methods are widely used for the microbiological analysis of human fluids or food samples.
- New methods also allow the semi-specific capture of microorganisms using functionalized cell surfaces. They use lectin or carbohydrate derivatives, peptides and peptide-mimicking compounds and are applied to the broad spectrum capture and / or specific binding of microorganisms in the sample.
- antimicrobial peptides linked to insoluble compounds have been used to kill, immobilize and detect microorganisms.
- polylysine is known for its antimicrobial activity.
- One of the aims of the invention is to provide functionalized glass beads capable of capturing microorganisms under simple conditions of use.
- Another object of the invention is to provide a simple process for preparing these glass beads functionalized with lysine or polylysine.
- Another object of the invention is to provide a device comprising the functionalized glass beads for use in the uptake of microorganisms.
- Another object of the invention is to provide an efficient method of capturing microorganisms for use in removing microorganisms or in diagnosis.
- a first object of the present invention is a glass bead functionalized with lysine or polylysine adsorbed on its surface.
- glass ball is understood to mean an element of spherical shape composed of glass.
- lysine is understood to mean the amino acid represented by the formula below.
- polylysine is understood to mean a polymer of lysine.
- the term “adsorbed” is understood to mean the fact that the lysine or polylysine molecules are attached to the surface of the glass beads by weak bonds of the Van der Waals type without modifying the molecular structure of the glass or of lysine or polylysine.
- the process for functionalizing the beads used in this invention involves adsorbing a polycation onto a negative surface such as glass.
- the adsorption process has the advantage of being easy to implement. The latter does not require any particular material or device unlike the implementation of a covalent bond between the polylysine and the glass beads.
- the invention constitutes a non-specific solution for the capture / elimination of microorganisms in a product without however altering it and then, for diagnostic purposes, to propose an elution solution allowing the release of all the microorganisms. retained on the column and ensuring cell viability. Only these two conditions will make it possible to provide a precise quantification of the living microorganisms contained in the sample analyzed.
- polylysine is a cationic polymer which improves cell adhesion and absorption of proteins or even nucleic acids by modifying the surface charges of the materials to which it is applied, for example certain plastics such as polystyrene or even glass.
- Polylysine surface treatments allow many applications including adhesion and spreading of various cell lines, cell differentiation and outgrowth of neurites, adhesion of transfected cell lines and survival of primary neurons in the cell. culture.
- the invention has many advantages over the techniques of the prior art. First of all, it makes it possible to avoid the use of heat treatment to eliminate microorganisms from potentially contaminated solutions and thus to preserve all the original qualities of the product without causing changes to its constituents.
- the invention also makes it possible to avoid using membrane filtration and its drawbacks linked, among other things, to clogging.
- the invention makes it possible to eliminate the microorganisms contained in liquid or liquid samples of large volume, samples which may be of different natures and have, for example, high turbidity or particles or sediments within them.
- the present invention relates in particular to a method for capturing and concentrating at least one microorganism likely to be present in a sample with a view to detecting it, quantifying it or evaluating its viability.
- a very low number of microorganisms is detectable which makes it possible to avoid carrying out an enrichment and thus to considerably reduce the analysis time but also to be able to quantify the initial target population.
- the invention solves the problems of mortality which can be induced by the use of polylysine in the context of a diagnosis and makes it possible to maintain the microorganism in its initial physiological state (dead or alive).
- the microorganism can here be a bacterium, a Fungi (for example a yeast or a mold) or a virus. This process is particularly applicable to microorganisms contained in complex media. These media corresponding to biological samples can be of human, food, cosmetic, veterinary or pharmaceutical origin.
- One of the major advantages of the present invention lies in the fact that the product does not undergo any modification on contact with the functionalized glass beads and that this also makes it possible to overcome problems of clogging.
- Another major advantage of the present invention is the possibility of using the device for capturing microorganisms in continuous flow, without enrichment steps, even if the quantity of microorganisms is low.
- this device allows a considerable saving of time over methods involving an enrichment step.
- the present invention relates to a glass bead functionalized by poly lysine adsorbed on its surface.
- the present invention relates to a glass bead as described above having a diameter of approximately 20 to approximately 1000 ⁇ m, in particular of approximately 20 to 30 ⁇ m, of approximately 30 to 40 ⁇ m, about 40 to 50 ⁇ m, about 50 to 75 ⁇ m, about 75 to 100 ⁇ m, about 100 to 200 ⁇ m, about 200 to 300 ⁇ m, about 300 to 400 ⁇ m, about 400 to 500 ⁇ m, about 500 to 600 ⁇ m, about 600 to 700 ⁇ m, about 700 to 800 ⁇ m, about 800 to 900 ⁇ m, about 900 to 1000 ⁇ m.
- the size of the glass beads is less than 20 ⁇ m, the beads pass through the frit intended to retain them. On the other hand, for beads larger than 1000 ⁇ m, the uptake rates obtained are low. The reduction in the size of the balls combined with a greater number of balls makes it possible to have a higher contact surface.
- the larger the size of the beads the greater the number of beads must be in order to maintain a constant uptake rate.
- the present invention relates to a glass bead as described above, in which the glass is of the soda-lime or borosilicate type.
- the type of glass used for the present invention especially soda-lime glass, has an advantage in terms of cost which is low.
- silica-lime-type glass is understood to mean a glass based on silica (Si0 2 ), calcium and sodium.
- borosilicate type glass means a glass based on silica (Si0 2 ) and on boron trioxide (B 2 0 3 ).
- the present invention relates to a glass bead as described above having a mass of approximately 10 ng to approximately 2 mg, in particular of approximately 10 to 50 ng, of approximately 50 to 100 ng, about 100 to 200 ng, about 200 to 500 ng, about 500 ng to 1 pg, about 1 to 5 pg, about 5 to 10 pg, about 10 to 20 pg, d 'about 20 to 50 pg, about 50 to 100 pg, about 100 to 200 pg, about 200 to 500 pg, about 500 pg to 1 mg, about 1 to 1.5 mg, about 1.5 to 2 mg.
- the present invention relates to a glass bead as described above, in which the lysine or polylysine has a molecular weight of approximately 146 to approximately 146,000 Da, in particular of approximately 146 to approximately 292 Da, from about 292 to about 1460 Da, from about 1460 to about 2920 Da, from about 2920 to about 4380 Da, from about 4380 to about 7300 Da, from about 7300 to about 14,600 Da, from about 14,600 to about 36,500 Da, about 36,500 to about 58,400 Da, about 58,400 to about 80,300 Da, about 80,300 Da to about 87,600 Da, about 87,600 Da to about 16,800 Da, around 116,800 to around 146,000 Da.
- the lysine or polylysine has a molecular weight of approximately 146 to approximately 146,000 Da, in particular of approximately 146 to approximately 292 Da, from about 292 to about 1460 Da, from about 1460 to about 2920 Da, from about 2920 to about 4380 Da, from about 4380 to
- the size of the polylysine can range from 292 to about 146,000 Da.
- the present invention relates to a glass bead as described above, in which the polylysine consists of a sequence of 2 to 1000 lysine units, in particular from 2 to 10, from 10 to 20, from 20 to 30, from 30 to 50, from 50 to 100, from 100 to 250, from 250 to 400, from 400 to 550, from 550 to 600, from 600 to 800, from 800 to 1000 lysine units.
- the present invention relates to a glass bead functionalized with lysine or polylysine in which the lysine or polylysine has a molecular weight of approximately 146 to approximately 80,300 Da, in particular of approximately 146 at about 292 Da, from about 292 to about 1460 Da, from about 1460 to about 2920 Da, from about 2920 to about 4380 Da, from about 4380 to about 7300 Da, from about 7300 to about 14,600 Da, from about 14,600 to about 36,500 Da, from about 36,500 to about 58,400 Da, from about 58,400 to about 80,300 Da.
- the size of the polylysine can range from 292 to about 80,300 Da.
- the present invention relates to a glass bead functionalized with lysine or polylysine in which the polylysine consists of a sequence of 2 to 550 lysine units, in particular from 2 to 10, from 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 250, 250 to 400, 400 to 550 lysine units.
- the present invention relates to a glass bead as described above, in which the lysine is L-lysine or D-lysine or a mixture of L-lysine and D-lysine , or the polylysine is of type a or e-poly-L-lysine or of type a or e-poly-D-lysine or a mixture of a or e-poly-L-lysine and a or e-poly-D- lysine, linear or branched, optionally in the form of a salt, in particular hydrobromide or hydrochloride.
- L-lysine and D-lysine is understood to mean the enantiomers represented by the formulas below.
- linear is understood to mean a polymer in which the sequence of the monomer units is carried out linearly.
- branched means a polymer in which the sequence of monomer units has branches.
- the present invention relates to a glass bead having a diameter of about 20 to about 1000 ⁇ m, and a mass of about 10 ng to about 2 mg, in which the glass is of the soda-lime or borosilicate type.
- lysine or polylysine functionalized by lysine or polylysine adsorbed to its surface, in which the lysine or polylysine has a molecular weight of about 146 to about 146,000 Da, is L-lysine or D-lysine or a mixture of L-lysine and D-lysine, or a or s-poly-L-lysine or a or s -poly-D -lysine or a mixture of a or e-poly-L-lysine and a or s -poly-D-lysine, linear or branched, optionally in the form of a salt, in particular hydrobromide or hydrochloride.
- a second object of the invention is a process for preparing a glass bead as described above comprising a step of bringing lysine or polylysine into contact with a glass bead to obtain a glass bead functionalized by lysine or polylysine adsorbed on its surface.
- the adsorption consists of contacting the polylysine at a concentration of 0.1% to 2% with the glass surface for an average period of about ten minutes. A shorter time has also been shown to allow adsorption of polylysine. With or without rinsing, the process makes it possible to effectively capture microorganisms. Rinsing with water nevertheless eliminates excess polylysine, reduces induced mortality and improves possible elution.
- the beads are functionalized and ready for use.
- the present invention relates to a preparation process as described above comprising a step of bringing lysine or polylysine into contact with a glass bead for a period of 1 minute to 24 hours, in particular 1 to 5 minutes, 5 to 10 minutes, 10 to 15 minutes, 15 to 20 minutes, 20 to 25 minutes, 25 to 30 minutes, 30 minutes to 1 hour, 1 to 2 hours, 2 to 5 hours, 5 to 10 hours, 10 to 24 hours, to obtain a glass bead functionalized with lysine or adsorbed polylysine.
- the present invention relates to a preparation process as described above comprising a step of bringing lysine or polylysine into contact with a glass bead for a period of 10 minutes, for obtain a glass bead functionalized with lysine or adsorbed polylysine.
- the present invention relates to a preparation process as described above comprising a drying step for a period of 1 minute to 12 hours, in particular from 1 to 5 minutes, from 5 to 10 minutes, 10 to 15 minutes, 15 to 20 minutes, 20 to 25 minutes, 25 to 30 minutes, 30 minutes to 1 hour, 1 to 2 hours, 2 to 5 hours, 5 to 12 hours, for obtain a glass bead functionalized with lysine or adsorbed polylysine.
- the present invention relates to a preparation process as described above comprising a drying step for a period of 10 minutes, to obtain a glass bead functionalized with lysine or polylysine adsorbed.
- the 10 min drying time corresponds to the duration of the drying step when the latter is accelerated using a vacuum chamber system with the vacuum set to its maximum (-900 mbar at the manometer before opening of the valve).
- drying means a method of drying in the open or accelerated using a vacuum chamber system, or by lyophilization.
- the present invention relates to a preparation process as described above in which the drying step is carried out in the open air or accelerated using a vacuum chamber system, or by lyophilization, in particular at a temperature ranging from room temperature to 60 ° C, in particular from room temperature to 30 ° C, from 30 ° C to 40 ° C, from 40 ° C to 50 ° C and from 50 ° C at 60 ° C.
- room temperature is meant a temperature ranging from about 20 ° C to about 25 ° C.
- the present invention relates to a preparation process as described above comprising a step of rinsing with an aqueous solution, in particular water, prior to the drying step, in order to remove the non-polylysine. adsorbed during the contacting step.
- aqueous solution means a liquid phase containing water.
- aqueous solutions that can be used are water, aqueous solutions of 150 mM NaCl, 200 mM NaCl and 0.01N HCl.
- the objective during the rinsing can be to lower the pH using an acid solution in order to maximize the positive charges on the surface of the polylysine, it is possible in this case to use acids such as acid citric or acetic acid.
- a third object of the invention is a device comprising a container containing glass beads as described above.
- This device can be used in static or in flow.
- the static mode makes it possible to increase the contact time between the glass beads functionalized by lysine or polylysine and the contaminated liquid and therefore to maximize the probability of encounter between the beads and the microorganisms. This probability of encounter can be further improved by carrying out manual or mechanical agitation.
- the disadvantage of this technique concerns large volumes of solution to be analyzed or to be debacterized (100 mL, 250 mL) since they require a large amount of beads, and therefore a higher hit than for the flow method.
- the flow method it is possible to pass large volumes in a small space to force, in a way, the microorganisms to meet the polylysine adsorbed from the beads (the quantity of beads used may be 1g) .
- the static method can be used for small volumes but not for large volumes.
- the term “container” means an object intended to receive the glass beads.
- the present invention relates to a device as described above in which the container is in the form of a tube, a flask, a beaker or a jar.
- the present invention relates to a device as described above, in which the container is in the form of a column optionally comprising a frit.
- the column and the frit can be two elements of the same set, or two separate elements which can be assembled.
- the present invention relates to a device as described above, in which the column has a volume of approximately 0.5 mL to approximately 1 L, in particular of approximately 0.1 mL to 1 mL. , about 1 mL to 2 mL, about 2 mL to 5 mL, about 5 mL to 10 mL, about 10 mL to 50 mL, about 50 mL to 100 mL, about 100 mL to 200 mL, about 200 mL to 500 mL, about 500 mL to 1 L, and the frit has a porosity less than the size of the glass beads used.
- the present invention relates to a device as described above, in which the total mass of the glass beads is approximately 10 mg to approximately 1 kg, in particular approximately 10 to 50 mg, d about 50 to 100 mg, about 100 to 200 mg, about 200 to 500 mg, about 500 mg to 1 g, about 1 to 2 g, about 2 to 5 g, of about 5 to 10 g, about 10 to 50 g, about 50 to 100 g, about 100 to 200 g, about 200 to 500 g, about 500 g to 1 kg.
- the present invention relates to a device as described above, comprising a container containing glass beads functionalized with lysine or polylysine adsorbed on its surface, said container being in particular in the form of a column.
- a frit optionally comprising a frit, said column having in particular a volume of approximately 0.5 mL to approximately 1 L, and said frit having in particular a porosity less than the size of the glass beads used.
- the present invention relates to a device as described above, in which the lysine or polylysine has a molecular weight of approximately 146 to approximately 146,000 Da, in particular of approximately 146 to approximately 292 Da. , from about 292 to about 1460 Da, from about 1460 to about 2920 Da, from about 2920 to about 4380 Da, from about 4380 to about 7300 Da, from about 7300 to about 14 600 Da , from about 14,600 to about 36,500 Da, from about 36,500 to about 58,400 Da, from about 58,400 to about 80,300 Da, from about 80,300 Da to about 87,600 Da, from about 87,600 Da to about 116,800 Da, from about 116,800 to about 146,000 Da, and / or in which the polylysine consists of a chain of 2 to 1000 lysine units, especially 2 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 250, 250 to 400, 400 to 550, 550 to 600,
- the present invention relates to a device as described above, in which the lysine or polylysine has a molecular weight of approximately 146 to approximately 80,300 Da, in particular of approximately 146 to approximately 292 Da , from about 292 to about 1460 Da, from about 1460 to about 2920 Da, from about 2920 to about 4380 Da, from about 4380 to about 7300 Da, from about 7300 to about 14 600 Da, from about 14,600 to about 36,500 Da, from about 36,500 to about 58,400 Da, from about 58,400 to about 80,300 Da, and / or in which the polylysine consists of a chain of 2 to 1000 lysine units, in particular from 2 to 10, from 10 to 20, from 20 to 30, from 30 to 50, from 50 to 100, from 100 to 250, from 250 to 400, from 400 to 550 lysine units.
- the polylysine consists of a chain of 2 to 1000 lysine units, in particular from 2 to 10, from 10 to 20, from 20 to 30, from 30
- the present invention relates to a device as described above, comprising glass beads having a diameter of about 20 to about 1000 ⁇ m, and / or having a mass of about 10 ng to about 2 mg, and / or in which the glass is of the soda-lime or borosilicate type.
- the present invention relates to a device as described above, in which the lysine is L-lysine or D-lysine or a mixture of L-lysine and D-lysine, or the polylysine is a or e-poly-L-lysine or a or e-poly-D-lysine or a mixture of a or s-poly-L-lysine and a or e-poly-D-lysine, linear or branched, optionally in the form of a salt, in particular hydrobromide or hydrochloride.
- the present invention relates to a device as described above, comprising a container containing glass beads as described above, said container being in particular in the form of a column optionally comprising a frit, said column having in particular a volume of about 0.5 mL to about 1 L, and said frit having in particular a porosity less than the size of the glass beads used.
- One of the first applications of the process is the elimination of microorganisms: thanks to capture, the sample is depleted of the microorganisms initially present.
- a second envisaged application is the diagnosis: the capture is then used as a means for concentrating the microorganisms for a diagnosis.
- the latter can be qualitative, of the presence / absence type, or quantitative. It may or may not be specific to the type of microorganism, and whether or not to differentiate living cells from dead cells.
- the analysis of the microorganisms captured can be done directly after capture on beads. These tests can for example be based on the detection of the whole cell or the detection / quantification of one of its constituents (DNA, RNA, ATP, enzymes and their activities or more broadly proteins, etc.). To do this, it is possible to elute the microorganisms or else to lyse them.
- cytometry flow or solid phase cytometry, colorimetry, spectroscopy, microscopy, ATPmetry (ATP: Adenosine triphosphate), PCR (Polymerase Chain Reaction or chain reaction by polymerase), RT-PCR (Reverse Transcriptase Polymerase Chain Reaction or polymerase chain reaction after reverse transcription), isothermal amplification or even immunological detection.
- PCR Polymerase Chain Reaction or chain reaction by polymerase
- RT-PCR Reverse Transcriptase Polymerase Chain Reaction or polymerase chain reaction after reverse transcription
- isothermal amplification isothermal amplification or even immunological detection.
- a fourth object of the invention is the use of glass beads as described above or of a device as described above, for the capture of microorganisms for the purpose of diagnosis.
- microorganism is understood to mean any prokaryotic or eukaryotic microscopic organism such as bacteria, yeasts, fungi and viruses. These microorganisms can be alive or dead. They are made up of DNA, a plasma membrane and different cell organelles. Red blood cells, presenting a plasma membrane, are excluded from this definition.
- the term “diagnostic” means the detection of the microorganisms captured.
- diagnostic techniques are in particular flow or solid phase cytometry, colorimetry, spectroscopy, microscopy, ATPmetry (ATP: Adenosine triphosphate), PCR (Polymerase Chain Reaction or chain reaction by polymerase), RT-PCR (Reverse Transcriptase Polymerase Chain Reaction or polymerase chain reaction after reverse transcription), isothermal amplification, or immunological detection.
- flow or solid phase cytometry colorimetry, spectroscopy, microscopy
- ATPmetry ATP: Adenosine triphosphate
- PCR Polymerase Chain Reaction or chain reaction by polymerase
- RT-PCR Reverse Transcriptase Polymerase Chain Reaction or polymerase chain reaction after reverse transcription
- isothermal amplification or immunological detection.
- the present invention relates to a use as described above, for the capture of microorganisms with a view to eliminating or reducing the charge of microorganisms from liquid or viscous samples. likely to contain said microorganisms.
- the term “elimination or reduction of the load of microorganisms” means the action of reducing the quantity of microorganisms present in a sample.
- the present invention relates to a use of glass beads functionalized with lysine or polylysine adsorbed on their surface, for the capture of microorganisms with a view to diagnosis, or with a view to lysine. elimination or reduction of the load of microorganisms from liquid or viscous samples liable to contain said microorganisms.
- the present invention relates to a use as described above of a device comprising glass beads functionalized with lysine or polylysine and as defined above, for the capture of microorganisms.
- a device comprising glass beads functionalized with lysine or polylysine and as defined above, for the capture of microorganisms.
- -organisms with a view to diagnosis, or with a view to eliminating or reducing the load of microorganisms from liquid or viscous samples liable to contain said microorganisms.
- the present invention relates to a use as described above, in which the lysine or polylysine has a molecular weight of approximately 146 to approximately 146,000 Da, in particular of approximately 146 to approximately 292 Da, about 292 to about 1460 Da, about 1460 to about 2920 Da, about 2920 to about 4380 Da, about 4380 to about 7300 Da, about 7300 to about 14,600 Da, about 14 600 to approximately 36,500 Da, approximately 36,500 to approximately 58,400 Da, approximately 58,400 to approximately 80,300 Da, approximately 80,300 Da to approximately 87,600 Da, approximately 87,600 Da to approximately 116 800 Da, from approximately 116,800 to approximately 146,000 Da, and / or in which the polylysine consists of a chain of 2 to 1000 lysine units, in particular from 2 to 10, from 10 to 20, from 20 to 30, from 30 to 50, from 50 to 100, from 100 to 250, from 250 to 400, from 400 to 550, from 550 to 600, from 600 to 800
- microorganisms for the capture of microorganisms for the purpose of diagnosis, or for the elimination or reduction of the load of microorganisms from liquid or viscous samples which may contain said microorganisms.
- the present invention relates to a use as described above, in which the lysine or polylysine has a molecular weight of approximately 146 to approximately 80,300 Da, in particular of approximately 146 to approximately 292 Da, from about 292 to about 1460 Da, from about 1460 to about 2920 Da, from about 2920 to about 4380 Da, from about 4380 to about 7300 Da, from about 7300 to about 14,600 Da, from about 14,600 to about 36,500 Da, about 36,500 to about 58,400 Da, about 58,400 to about 80,300 Da, and / or in which the polylysine consists of a sequence of 2 to 1000 lysine units, especially 2 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 250, 250 to 400, 400 to 550 lysine units ,
- microorganisms for the capture of microorganisms for the purpose of diagnosis, or for the elimination or reduction of the load of microorganisms from liquid or viscous samples which may contain said microorganisms.
- the present invention relates to a use as described above of glass beads as described above, said glass beads having a diameter of from about 20 to about 1000 ⁇ m, and / or having a diameter. mass of about 10 ng to about 2 mg, and / or in which the glass is of the soda-lime or borosilicate type, for the uptake of microorganisms with a view to diagnosis, or with a view to elimination or reduction the charge of microorganisms of liquid or viscous samples liable to contain said microorganisms.
- the present invention relates to a use as described above of glass beads as described above, in which the lysine is L-lysine or D-lysine or a mixture of L -lysine and D-lysine, or polylysine is a or e-poly-L-lysine or a or e-poly-D-lysine or a mixture of a or e-poly-L-lysine and a or e-poly-D-lysine, linear or branched, optionally in the form of a salt, in particular hydrobromide or hydrochloride, for the capture of microorganisms with a view to diagnosis, or with a view to the elimination or the reduction in the load of microorganisms in liquid or viscous samples liable to contain said microorganisms.
- a salt in particular hydrobromide or hydrochloride
- the present invention relates to a use as described above in which said glass beads are defined above, for the capture of microorganisms with a view to diagnosis, or with a view to the elimination. or the reduction in the load of microorganisms in liquid or viscous samples liable to contain said microorganisms.
- Another subject of the invention is a method for capturing microorganisms comprising a step of bringing a liquid or viscous sample containing said microorganisms into contact with glass beads as described above or d a device as described above, under conditions making it possible to create an interaction between said microorganisms and the glass beads, and to obtain said microorganisms captured on the glass beads.
- the present invention relates to a method as described above, in which the proportion of microorganisms originating from the sample and captured on the glass beads is from 0.001% to 100% for the purpose of removal. microorganisms from said sample.
- the present invention relates to a method as described above, comprising an additional step of eluting the microorganisms previously captured under conditions allowing the separation of the aforementioned microorganisms captured from the aforesaid glass beads. and the recovery of said microorganisms.
- the present invention relates to a method as described above, comprising a step of bringing a liquid or viscous sample containing said microorganisms into contact with glass beads functionalized with water. lysine or polylysine, under conditions making it possible to create an interaction between said microorganisms and the glass beads, and to obtain said microorganisms captured on the glass beads, in particular in which the proportion of microorganisms from the sample and captured on the glass beads is from 0.001% to 100% for the elimination of microorganisms from said sample.
- the present invention relates to a method as described above, comprising a step of bringing a liquid or viscous sample containing said microorganisms into contact with a device comprising functionalized glass beads. with lysine or polylysine and as defined above, under conditions making it possible to create an interaction between said microorganisms and the glass beads, and to obtain said microorganisms captured on the glass beads , in particular in which the proportion of microorganisms originating from the sample and captured on the glass beads is from 0.001% to 100% for the purpose of eliminating microorganisms from said sample.
- the present invention relates to a process as described above, in which the glass beads are functionalized with lysine or polylysine having a molecular weight of approximately 146 to approximately 14,000 Da, especially from about 146 to about 292 Da, from about 292 to about 1460 Da, from about 1460 to about 2920 Da, from about 2920 to about 4380 Da, from about 4380 to about 7 300 Da, about 7,300 to about 14,600 Da, about 14,600 to about 36,500 Da, about 36,500 to about 58,400 Da, about 58,400 to about 80,300 Da, about 80 300 Da to about 87,600 Da, from about 87,600 Da to about 116,800 Da, from about 116,800 to about 146,000 Da, and / or in which the polylysine consists of a chain of 2 to 1000 lysine units , in particular from 2 to 10, from 10 to 20, from 20 to 30, from 30 to 50, from 50 to 100, from 100 to 250, from 250 to 400, from 400 to 550
- the glass is of the soda-lime or borosilicate type, for the capture of microorganisms for the purpose of diagnosis, or for the elimination or reduction of the load of microorganisms from liquid or viscous samples liable to contain said microorganisms.
- the present invention relates to a process as described above, in which the lysine is L-lysine or D-lysine or a mixture of L-lysine and D-lysine, or the polylysine is a or e-poly-L-lysine or a or e-poly-D-lysine or a mixture of a or s-poly-L-lysine and a or e-poly-D-lysine, linear or branched, optionally in the form of a salt, in particular hydrobromide or hydrochloride, for the capture of microorganisms with a view to diagnosis, or with a view to eliminating or reducing the load of microorganisms d liquid or viscous samples likely to contain said microorganisms.
- a salt in particular hydrobromide or hydrochloride
- the present invention relates to a process as described above, in which the lysine or polylysine has a molecular weight of approximately 146 to approximately 80,300 Da, in particular of approximately 146 to approximately 292 Da , from about 292 to about 1460 Da, from about 1460 to about 2920 Da, from about 2920 to about 4380 Da, from about 4380 to about 7300 Da, from about 7300 to about 14,600 Da, from about 14,600 to about 36,500 Da, from about 36,500 to about 58,400 Da, from about 58,400 to about 80,300 Da, and / or in which the polylysine consists of a chain from 2 to 1000 lysine units, in particular from 2 to 10, from 10 to 20, from 20 to 30, from 30 to 50, from 50 to 100, from 100 to 250, from 250 to 400, from 400 to 550 lysine units,
- the present invention relates to a method as described above, comprising glass beads functionalized with lysine or polylysine defined above, comprising a step of bringing a sample into contact. liquid or viscous containing said microorganisms, with a device as described above, under conditions making it possible to create an interaction between said microorganisms and the glass beads, and to obtain said microorganisms captured on the glass beads, in particular in which the proportion of microorganisms originating from the sample and captured on the glass beads is from 0.001% to 100% for the purpose of eliminating microorganisms from said sample.
- the solution in question must remain in contact with the glass beads on which the microorganisms have been captured for a minimum period of 5 to 40 minutes, in particular 5 to 10, 10 to 15, 15 to 20, 20 to 30, and 30 to 40 minutes.
- this step is carried out at a temperature which may range from 20 to 55 ° C., preferably under the optimum temperature conditions of the enzyme used.
- this step is carried out at room temperature.
- the present invention relates to a method as described above, in which, during the elution step, the proportion of microorganisms separated from the glass beads on which the aforesaid microorganisms had been previously captured and then recovered is from 0.001% to 100%.
- the present invention relates to a method as described above, in which the recovery of the microorganisms is carried out with a view to a diagnosis.
- This diagnosis can be qualitative, of the presence / absence type, or quantitative. It may or may not be specific to the type of microorganism, and may or may not differentiate living cells from dead cells.
- the present invention relates to a method as described above, comprising:
- the present invention relates to a method as described above, in which the elution step is carried out using an elution solution of enzymatic or chemical type.
- the enzymatic-type elution solutions that can be used are, for example, trypsin, accumulax (ACCUMAX enzymes in Dulbecco PBS (0.2 g / L KC1, 0.2 g / L KH 2 P0 4 , 8 g / L NaCl, and 1.15 g / L Na 2 HP0 4 )).
- accutase (Accutase enzymes in Dulbecco PBS (0.2 g / L KC1, 0.2 g / L KH 2 P0 4 , 8 g / L NaCl, and 1.15 g / L Na 2 HP0 4 ) containing 0.5 mM EDTA 4Na and 3 mg / L of Phenol Red).
- the chemical-type elution solutions that can be used are for example: IM NaCl, EDTA 0.1 to 10%, sodium bicarbonate 1%, sodium citrate 10%, acetic acid 0.1 to 10%, methanol 0.1 to 10%, pluronic F-127 0.01 to 0.1% (poloxamer 407, nonionic three-block copolymer: Poly (ethylene glycol) -block-poly (propylene glycol) -block-poly (ethylene glycol)).
- the present invention relates to a method as described above, in which the bringing of the sample into contact with the glass beads or the device takes place statically or in flow.
- the term “statically” is understood to mean the fact that the contacting between the sample and the glass beads takes place in a container without there being a continuous movement of the sample and that the sample is renewed during contacting.
- the static contacting requires that at the end of the contact time the sample is separated from the glass beads. This step can for example be carried out by sucking the liquid through a frit or by removing the liquid.
- the step of bringing the sample containing the microorganisms into contact with glass beads is carried out over a period of 5 to 15 minutes before the aspiration is triggered and carried out using the vacuum chamber.
- the term “in flow” is understood to mean the fact that the bringing into contact between the sample and the glass beads takes place in a container making it possible to cause the sample to flow continuously between them.
- the present invention relates to a method as described above, in which the microorganisms are chosen from bacteria, and Fungi, in particular yeasts and fungi.
- the present invention relates to a method as described above, in which the Fungi belong in particular to the genera Absidia, Alternaria, Aspergillus, Aureobasidium, Botrytis, Brettanomyces, Byssochlamys, Candida, Chaetomium, Cladosporium, Colletotrichum, Cryptococcus, Debaryomyces, Emericella, Epicoccum, Eupenicillium, Eurotium, Fusarium, Galactomyces, Geotrichum, Gliocladium, Hanseniaspora, Humicola, Hyphopichia, Kluyveromyces, Lichtheimia, Lodderomyces, Meyerozyma, Monascosestora, Pucadicillomyces, Myascénomyces,
- the present invention relates to a method as described above, in which the bacteria are Gram + or Gram bacteria.
- the present invention relates to a method as described above, in which the bacteria belong in particular to the genera Acetobacter, Achromobacter, Acidovorax, Acinetobacter, Actinomyces, Aerococcus, Aeromonas, Alcaligenes, Alicyclobacillus, Aquaspirillum, Asaia, Bacillus, Bifidobacterium sp., Bordetella, Brachybacterium, Brevibacillus, Brevibacterium, Brevundimonas, Burkholderia, Buttiauxella, Campylobacter, Carnobacterium, Cellulomona, Citrobacter, Clavibacter Clostridium, Corynebacterium, Cronobacteria, Cupobriacidacteria, Enterlamocacteria, Escapeylobacteria, Enterlamackacterium, Enterlamocacteria, Elizabeth, Enteroccichacterium, Enterlamocacteria, Eskococcinacterium, Enter
- the present invention relates to a method as described above, in which the liquid or viscous sample is chosen from:
- a biological sample such as burin, blood, synovial fluid, lymph, tear fluid, secretions, mucous membranes, - a pharmaceutical sample, such as injectable solutions, syrups, vaccines, eye drops, ophthalmic gels,
- a cosmetic sample such as make-up removers, products for cleaning the skin, deodorants, products intended for shaving, self-tanners, sun protection creams, solvents, shampoos, conditioners,
- a food sample such as drinks, in particular water (still, sparkling and / or flavored), milk, fruit juices, sodas, alcoholic beverages, tea-based drinks, meats, ready meals, dairy products, egg products.
- biological sample is understood to mean a sample originating from a body fluid or from a tissue of a human or animal body.
- biological samples examples include urine, blood, synovial fluid, lymph, tear fluid, secretions, mucous membranes.
- the term "pharmaceutical sample” means a sample from a pharmaceutical product containing chemical, natural or synthetic substances for human or veterinary use.
- Examples of pharmaceutical samples which can be used are injectable solutions, syrups, vaccines, eye drops, ophthalmic gels.
- the term “cosmetic sample” is understood to mean a sample originating from a cosmetic product containing a substance or a mixture intended to be brought into contact with the various surface parts of the human body, in view, exclusively or mainly. , to clean them, to perfume them, to modify their appearance, to protect them, to keep them in good condition or to correct body odor.
- cosmetic samples which can be used are makeup removers, skin cleansers, deodorants, shaving products, self-tanners, sunscreen creams, solvents, shampoos, conditioners.
- the term "food sample” means a sample from a food product which can be used as food or drink for a human or animal.
- Examples of food samples that can be used are beverages including water (still, sparkling and / or flavored), milk, fruit juices, sodas, alcoholic beverages, tea-based beverages, meats, ready meals, dairy products, egg products.
- Another object of the invention is a kit comprising glass beads, lysine or polylysine and optionally a device in the form of a column and elution solutions of enzymatic or chemical type, for the capture of microorganisms. with a view to diagnosis, or with a view to eliminating or reducing the load of microorganisms from liquid or viscous samples liable to contain said microorganisms.
- Figure IA General operating principle of capture / elimination of microorganisms in flow mode
- FIG. 1A represents the general operating principle of the capture / elimination of microorganisms in flow mode.
- 1 represents the addition of the potentially contaminated solution
- 2 represents the column
- 3 represents the glass beads functionalized using lysine or polylysine
- 4 represents the frit
- 5 represents the solution devoid of its microorganisms at the outlet of the column
- 6 represents an enlargement of the microorganisms immobilized in contact with the functionalized beads.
- Figure IB General operating principle of flow mode elution
- 1 represents the addition of the eluting solution
- 2 represents the elution solution at the outlet of the column containing the previously immobilized microorganisms.
- Figure 2A General operating principle for capturing / eliminating microorganisms in static mode
- FIG. 2A represents the general principle of operation of the capture / elimination of microorganisms in static mode.
- 1 represents the addition of the potentially contaminated solution
- 2 represents the container
- 3 represents the potentially contaminated solution brought into contact with the glass beads
- 4 represents glass beads functionalized with lysine or polylysine
- 5 represents an enlargement of the microorganisms immobilized in contact with the functionalized beads
- 6 represents the recovery or disposal of the solution devoid of its microorganisms.
- Figure 2B General operating principle of elution in static mode 1 represents the addition of the elution solution; 2 represents the sampling of the elution solution provided with the eluted microorganisms.
- Example 1 General protocol for adsorption of polv-1-lvsine on glass beads
- the poly-L-lysine solution was aspirated under vacuum before rinsing with 6 mL (6 x 1 mL) of deionized water was performed for each column.
- the vacuum pump was set to 700 mbar of vacuum. After rinsing, the columns are functionalized and ready for use.
- Strains of microorganisms were thawed using a cryobead in an appropriate liquid or solid medium before being placed at a temperature that allowed them to grow for the time required for their growth. Strains were subcultured at least two times at 2% in an appropriate liquid medium or by transfer to solid medium before being used for testing.
- a small volume (500 ⁇ L to 1 mL) of solution containing a given quantity of microorganisms (20 to ⁇ 10 7 units) was introduced into a column containing 1 g of glass beads functionalized with poly-1-lysine.
- a contact time of 5 to 15 min was applied before the suction was triggered and carried out using the vacuum chamber, the vacuum of the pump being set at 50 mbar.
- a given volume (from 500 ⁇ L to 100 mL) of solution containing a given quantity of microorganisms (from 20 to ⁇ 10 7 units) was filtered through a column containing 1 g of glass beads functionalized with poly-1 -lysine .
- the aspiration was triggered before the introduction of the solution and was carried out using the vacuum chamber, the vacuum of the pump was set at 50 mbar.
- an elution solution was introduced in order to remove the microorganisms.
- the amount of elution solution was variable but was at least 500 pL in order to cover all the beads present in the column.
- the solution was (1) enzymatic, (2) chemical and / or (3) both applied as a mixture or successively.
- Example 6-1 General enzymatic elution protocol
- the enzymatic solutions were purchased ready to use, the concentration may vary (eg: Trypsin 0.05% to 2.5%).
- the aspiration was triggered after a more or less long contact time (minimum 5 min, the longest time tested is 40 min) in order to allow time for the enzymes to act.
- the incubation temperature applied was from room temperature to 42 ° C depending on the enzyme solution that was used. It was for example possible to make the trypsin act at room temperature or at 42 ° C (37 ° C being the optimum temperature).
- the filtration was then carried out using the vacuum chamber, the vacuum of the pump was set at 700 mbar.
- Example 6-2 General chemical elution protocol All chemical solutions (IM NaCl, EDTA 0.1 to 10%, sodium bicarbonate 1%, sodium citrate 10%, acetic acid 0.1 to 10%, methanol 0.1 to 10%, pluronic F-127 0.01 to 0.1%) were prepared. using deionized water and then filtered through a 0.2 ⁇ m filter. The percentages mentioned above correspond to a weight / volume ratio of water (g / 100 mL). The aspiration was triggered before introduction of the solution or after a more or less important contact time (5 minutes minimum tested). Filtration was carried out using the vacuum chamber, the vacuum of the pump being set at 700 mbar.
- Example 6-3 General enzymatic and / or chemical elution protocol mixed or successively
- the two types of enzymatic and chemical solutions could also be applied as a mixture (eg: Trypsin-EDTA) or successively (eg: Accumax followed by NaCl). It was necessary to respect a mandatory contact time for the enzyme solutions in order, once again, to allow the enzymes to act.
- Example 7 General protocol for evaluating the uptake and / or elution rate
- the evaluation of the capture or elution rate could be carried out in different ways depending on the amount of microorganisms present and the volume of solution to be analyzed.
- Example 7-1 Evaluation of the uptake and / or elution rate by flow cytometry
- the flow cytometry required a relatively high concentration of microorganism to obtain a reliable result ( ⁇ 10 5 CFU / mL).
- the results in terms of uptake and elution rate were obtained as described below.
- the permeates obtained after capture were analyzed using a cytometer to assess the number of microorganisms not captured. It was thus possible to deduce the number of microorganisms captured on the column given that the quantity of microorganisms introduced beforehand into the column is known (count carried out with a cytometer). For the elution, the number of microorganisms eluted was directly measured using the cytometer.
- Example 7-2 Evaluation of the rate of uptake and / or elution by filtration on a membrane deposited on agar medium
- Membrane filtration made it possible to concentrate the microorganisms. It was then possible to deposit this membrane on a medium favorable to their growth at a given temperature and for a defined time.
- the independent filtrations of the permeates and elutes thus made it possible to calculate the uptake and elution rates in the same way as for flow cytometry. For this it was necessary to count the colonies on the membrane after the incubation time.
- Example 7-3 Evaluation of the uptake and / or elution rate by membrane filtration for observation under a microscope
- the membrane filtration made it possible to concentrate the microorganisms and to observe them after labeling directly under a microscope. In the same way as before, it was then necessary to count the microorganisms present on the membrane using a microscope.
- the markers make it possible to describe the physiological state of microorganisms (living or dead) at a given time, which growth on a membrane in a Petri dish (Example 7-2) or spreading on agar medium does not allow (Example 7-2) or spreading on agar medium ( Example 7-4) for which only viable and cultivable microorganisms are observable, at least 24 hours after deposition.
- Example 7-4 Evaluation of the uptake and / or elution rate by membrane filtration for spreading on a Petri dish
- Another method consists of spreading all or part of the permeates and eluates on an agar medium (in a Petri dish) favorable to the growth of microorganisms. This method required a variable incubation time at a given temperature. In the same way as before, it was then necessary to count the microorganisms present on the box in order to deduce the capture and elution rates, the number of microorganisms introduced being known using the same spreading method.
- Fes 500 pF of suspension which had passed through the column were collected in a tube and then a count was carried out by successive dilutions to tenths in physiological water 0.85% NaCl, by spreading on agar media in a Petri dish. The same microorganisms were tested at high and low concentrations.
- Table 1 shows the number of Colony Forming Units introduced and the corresponding uptake rate.
- Table 2 shows the number of Colony Forming Units introduced and the corresponding uptake rate.
- Table 3 shows the number of Colony Forming Units introduced and the corresponding uptake rate.
- a count of the mother suspension of E. coli CIP 54.8 tested was carried out by successive dilutions to tenths in physiological water 0.85% NaCl and spreading on agar media in a Petri dish.
- 500 ⁇ L of suspension at the desired concentration were added per column and sucked as a flow through the column using the vacuum chamber (50 mbar) and pump system.
- a count of the permeates was carried out by successive dilutions to the tenth in physiological water 0.85% NaCl and spreading on agar media in a Petri dish.
- Table 5 shows the column and the corresponding elution rate.
- Elution protocol 2 successive eludons with 0.05% trypsin - EDTA IX (in PBS without Calcium, without Magnesium, with Phenol Red) of 500 ⁇ L each were carried out per column.
- the first elution was carried out respecting a contact time of 15 min at 37 ° C.
- the following was carried out in a flow without prolonged contact time of the solution and at 50 mbar of vacuum.
- the eluates were collected in a tube using the vacuum chamber system.
- Table 6 shows the number of Colony Forming Units introduced and the corresponding uptake rate.
- Table 7 shows the column, the microorganism and the corresponding elution rate.
- Micro-organism capture / elimination protocol First, a count of the mother suspension of Staphylococcus aureus tested was carried out by successive dilutions to tenths in physiological water 0.85% NaCl, by cytometry. 500 ⁇ L of suspension at the desired concentration were added per column and sucked as a flow through the column using the vacuum chamber (50 mbar) and pump system. A count of the permeates was carried out by cytometry.
- Table 8 shows the number of Colony Forming Units introduced and the corresponding uptake rate.
- Table 9 shows the column, the microorganism and the corresponding elution rate.
- Elution 1 Sodium citrate 1% - EDTA 0.1% (3.5 mL) then physiological water 0.85% NaCl (0.5 mL)
- Elution 2 Trypsin 2.5% (0.5 mL, 30 min at room temperature)
- the aspiration was carried out at 50 mbar of vacuum.
- the eluates were collected in different tubes using the vacuum chamber system.
- Table 10 shows the number of Colony Forming Units introduced and the corresponding uptake rate.
- Table 11 shows the column, the microorganism and the corresponding elution rate.
- 300 mg of glass beads with a diameter of 30-50 mih are distributed in 0.8 mL columns. 142.8 ⁇ L of 0.5% (w / v) poly-L-lysine (10 lysines per poly-L-lysine chain) are added to each column. A contact time of 5 min is observed between the beads and the polylysine.
- the solution is then removed using a vacuum chamber system and a pump set to 50 mbar of vacuum. Rinsing of the column is carried out using 1 mL of molecular biology water with the same depression. The pump is then set to 700 mbar of vacuum for drying for 5 min. After this step, the poly-L-lysine functionalized glass beads are ready for use.
- the stock suspension of Fusarium oxysporum (UBOCC-A-112042) was diluted to 10 th in physiological saline 0.85% NaCl and then enumerated by flow cytometry. 500 ⁇ L of suspension at the desired concentration were added per column and sucked as a flow through the column using the vacuum chamber (50 mbar) and pump system. A count of the permeates was carried out by cytometry.
- Table 12 shows the number of Colony Forming Units introduced and the corresponding uptake rate.
- Table 13 shows the column, the microorganism and the corresponding elution rate.
- Example 16 fungus Mucor racemosus
- Mother suspension Mucor racemosus (ATCC 42647) was diluted to 10 th in physiological saline 0.85% NaCl and then enumerated by flow cytometry. 500 ⁇ L of suspension at the desired concentration were added per column and sucked as a flow through the column using the vacuum chamber (50 mbar) and pump system. A count of the permeates was carried out by cytometry.
- Table 14 shows the number of Colony Forming Units introduced and the corresponding uptake rate.
- Table 15 shows the column, the microorganism and the corresponding elution rate.
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Pathology (AREA)
- General Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Inorganic Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR1902972A FR3094016B1 (fr) | 2019-03-22 | 2019-03-22 | Billes de verre fonctionnalisées, leur utilisation pour capter des micro-organismes et les dispositifs correspondants. |
PCT/EP2020/057162 WO2020193260A1 (fr) | 2019-03-22 | 2020-03-16 | Billes de verre fonctionnalisees, leur utilisation pour capter des micro-organismes et les dispositifs correspondants |
Publications (1)
Publication Number | Publication Date |
---|---|
EP3942034A1 true EP3942034A1 (fr) | 2022-01-26 |
Family
ID=67262670
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP20710190.8A Pending EP3942034A1 (fr) | 2019-03-22 | 2020-03-16 | Billes de verre fonctionnalisees, leur utilisation pour capter des micro-organismes et les dispositifs correspondants |
Country Status (5)
Country | Link |
---|---|
US (1) | US20220186282A1 (fr) |
EP (1) | EP3942034A1 (fr) |
CA (1) | CA3134894A1 (fr) |
FR (1) | FR3094016B1 (fr) |
WO (1) | WO2020193260A1 (fr) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112980977B (zh) * | 2021-03-19 | 2022-11-25 | 天水师范学院 | 苹果树枝干腐烂病的致病菌鉴别引物及患病程度分级方法 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5534416A (en) * | 1993-04-13 | 1996-07-09 | Molecular Probes, Inc. | Fluorescent viability assay using cyclic-substituted unsymmetrical cyanine dyes |
US9766237B2 (en) * | 2013-03-15 | 2017-09-19 | Iris International, Inc. | Method of capturing bacteria on polylysine-coated microspheres |
-
2019
- 2019-03-22 FR FR1902972A patent/FR3094016B1/fr active Active
-
2020
- 2020-03-16 CA CA3134894A patent/CA3134894A1/fr active Pending
- 2020-03-16 US US17/441,902 patent/US20220186282A1/en active Pending
- 2020-03-16 WO PCT/EP2020/057162 patent/WO2020193260A1/fr active Application Filing
- 2020-03-16 EP EP20710190.8A patent/EP3942034A1/fr active Pending
Also Published As
Publication number | Publication date |
---|---|
WO2020193260A1 (fr) | 2020-10-01 |
CA3134894A1 (fr) | 2020-10-01 |
FR3094016B1 (fr) | 2024-04-12 |
US20220186282A1 (en) | 2022-06-16 |
FR3094016A1 (fr) | 2020-09-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9932620B2 (en) | Methods, devices, and systems of detecting microorganisms | |
EP2356210B1 (fr) | Systeme automatise de lyse de microorganismes presents dans un echantillon, d'extraction et de purification des acides nucleiques desdits microorganismes aux fins d'analyse | |
EP1983343B1 (fr) | Utilisation d'un dispositif de concentration et de détection de germes pathogènes à partir de produits sanguins et/ou de leurs dérivés | |
EP2948548B2 (fr) | Procédé d'isolement spécifique d'acides nucléiques d'intérêt | |
EP2152850B1 (fr) | Dispositif de lyse de microorganismes presents dans un echantillon environnemental ou clinique et d'extraction des acides nucleiques desdits microorganismes aux fins d'analyse | |
FR2926560A1 (fr) | Procede d'extraction et de purification d'acides nucleiques sur membrane | |
US10774300B2 (en) | Methods and kits for isolating microorganisms from culture | |
US20130309700A1 (en) | Methods, devices, and systems of detecting microorganisms | |
WO2013084772A1 (fr) | Méthode de mesure de cellules et réactif pour la mesure des cellules | |
Zhang et al. | Bacteriocin assisted food functional membrane for simultaneous exclusion and inactivation of Alicyclobacillus acidoterrestris in apple juice | |
WO2010007255A2 (fr) | Procede de purification des acides nucleiques de microorganismes presents dans des echantillons liquides | |
EP2872867B1 (fr) | Systeme automatise de lyse de microorganismes presents dans un echantillon, d'extraction et de purification des acides nucleiques desdites microorganismes aux fins d'analyse | |
EP3942034A1 (fr) | Billes de verre fonctionnalisees, leur utilisation pour capter des micro-organismes et les dispositifs correspondants | |
EP1565568A1 (fr) | PROCEDE DE DETECTION UNIVERSELLE DE MICROORGANISMES ET MILIEU REACTIONNEL PERMETTANT LA MISE EN &OElig;UVRE DU PROCEDE | |
EP4052014A1 (fr) | Dispositif contenant des billes de verre fonctionnalisees avec du polyethyleneimine, et leur utilisation pour capter les micro-organismes | |
FR2915208A1 (fr) | Composition comprenant l'association d'edta et de pei pour augmenter la permeabilite des parois des microorganismes et utilisations de ladite composition | |
US20150140583A1 (en) | Methods, devices, and systems of detecting microorganisms | |
FR3082851A1 (fr) | Procede de detection et/ou d'identification d'au moins un microorganisme cible present sur une surface. |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: UNKNOWN |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE |
|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE |
|
17P | Request for examination filed |
Effective date: 20211020 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
DAV | Request for validation of the european patent (deleted) | ||
DAX | Request for extension of the european patent (deleted) | ||
P01 | Opt-out of the competence of the unified patent court (upc) registered |
Effective date: 20230517 |