CN110042142A - 一种用于检测大肠杆菌o157与非o157大肠杆菌的显色培养基 - Google Patents
一种用于检测大肠杆菌o157与非o157大肠杆菌的显色培养基 Download PDFInfo
- Publication number
- CN110042142A CN110042142A CN201811457345.4A CN201811457345A CN110042142A CN 110042142 A CN110042142 A CN 110042142A CN 201811457345 A CN201811457345 A CN 201811457345A CN 110042142 A CN110042142 A CN 110042142A
- Authority
- CN
- China
- Prior art keywords
- escherichia coli
- culture medium
- chromogenic
- coli
- detecting
- 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
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 138
- 239000001963 growth medium Substances 0.000 title claims abstract description 64
- 241001333951 Escherichia coli O157 Species 0.000 title claims abstract description 31
- 229930182470 glycoside Natural products 0.000 claims abstract description 39
- 239000003593 chromogenic compound Substances 0.000 claims abstract description 27
- 239000000758 substrate Substances 0.000 claims abstract description 21
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 19
- UPSFMJHZUCSEHU-JYGUBCOQSA-N n-[(2s,3r,4r,5s,6r)-2-[(2r,3s,4r,5r,6s)-5-acetamido-4-hydroxy-2-(hydroxymethyl)-6-(4-methyl-2-oxochromen-7-yl)oxyoxan-3-yl]oxy-4,5-dihydroxy-6-(hydroxymethyl)oxan-3-yl]acetamide Chemical compound CC(=O)N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@H]1[C@H](O)[C@@H](NC(C)=O)[C@H](OC=2C=C3OC(=O)C=C(C)C3=CC=2)O[C@@H]1CO UPSFMJHZUCSEHU-JYGUBCOQSA-N 0.000 claims abstract description 19
- 102000053187 Glucuronidase Human genes 0.000 claims abstract description 10
- 108010060309 Glucuronidase Proteins 0.000 claims abstract description 10
- -1 β-N-acetylglucosamine glycosides Chemical class 0.000 claims abstract description 10
- 239000003086 colorant Substances 0.000 claims abstract description 6
- MIAKOEWBCMPCQR-YBXAARCKSA-N (2s,3r,4s,5r,6r)-2-(4-aminophenoxy)-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound C1=CC(N)=CC=C1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MIAKOEWBCMPCQR-YBXAARCKSA-N 0.000 claims abstract description 3
- 102000006995 beta-Glucosidase Human genes 0.000 claims abstract description 3
- 108010047754 beta-Glucosidase Proteins 0.000 claims abstract description 3
- IOJUPLGTWVMSFF-UHFFFAOYSA-N benzothiazole Chemical compound C1=CC=C2SC=NC2=C1 IOJUPLGTWVMSFF-UHFFFAOYSA-N 0.000 claims description 76
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- 239000002253 acid Substances 0.000 claims description 18
- 150000002338 glycosides Chemical class 0.000 claims description 18
- 229920001817 Agar Polymers 0.000 claims description 16
- 239000008272 agar Substances 0.000 claims description 16
- 150000002989 phenols Chemical class 0.000 claims description 10
- 239000001888 Peptone Substances 0.000 claims description 9
- 108010080698 Peptones Proteins 0.000 claims description 9
- 235000019319 peptone Nutrition 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 229940041514 candida albicans extract Drugs 0.000 claims description 8
- 239000012138 yeast extract Substances 0.000 claims description 8
- 229940099352 cholate Drugs 0.000 claims description 6
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 claims description 6
- 229930182478 glucoside Natural products 0.000 claims description 6
- 150000008131 glucosides Chemical class 0.000 claims description 6
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 5
- PCKPVGOLPKLUHR-UHFFFAOYSA-N OH-Indolxyl Natural products C1=CC=C2C(O)=CNC2=C1 PCKPVGOLPKLUHR-UHFFFAOYSA-N 0.000 claims description 5
- NGFMICBWJRZIBI-JZRPKSSGSA-N Salicin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O1)c1c(CO)cccc1 NGFMICBWJRZIBI-JZRPKSSGSA-N 0.000 claims description 5
- NGFMICBWJRZIBI-UHFFFAOYSA-N alpha-salicin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UHFFFAOYSA-N 0.000 claims description 5
- 239000008101 lactose Substances 0.000 claims description 5
- NGFMICBWJRZIBI-UJPOAAIJSA-N salicin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UJPOAAIJSA-N 0.000 claims description 5
- 229940120668 salicin Drugs 0.000 claims description 5
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 5
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 3
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 claims description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 3
- 229920000053 polysorbate 80 Polymers 0.000 claims description 3
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 claims description 2
- 150000007942 carboxylates Chemical group 0.000 claims description 2
- 150000001732 carboxylic acid derivatives Chemical group 0.000 claims description 2
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims 3
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 claims 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims 1
- 239000005864 Sulphur Substances 0.000 claims 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 claims 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims 1
- 239000008103 glucose Substances 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 16
- 238000000926 separation method Methods 0.000 description 54
- 239000002585 base Substances 0.000 description 32
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 27
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 24
- 241000894006 Bacteria Species 0.000 description 20
- 241001135265 Cronobacter sakazakii Species 0.000 description 20
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 18
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 16
- 241000193830 Bacillus <bacterium> Species 0.000 description 14
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 14
- 206010017903 Gastroenteritis Escherichia coli Diseases 0.000 description 14
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 13
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 12
- WMFOQBRAJBCJND-UHFFFAOYSA-M Lithium hydroxide Chemical compound [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 229910052938 sodium sulfate Inorganic materials 0.000 description 9
- 235000011152 sodium sulphate Nutrition 0.000 description 9
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 239000002904 solvent Substances 0.000 description 8
- 241000193468 Clostridium perfringens Species 0.000 description 7
- 241000588697 Enterobacter cloacae Species 0.000 description 7
- 241000293871 Salmonella enterica subsp. enterica serovar Typhi Species 0.000 description 7
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 7
- 125000006239 protecting group Chemical group 0.000 description 7
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 6
- 238000005160 1H NMR spectroscopy Methods 0.000 description 6
- 241000301512 Bacillus cereus ATCC 14579 Species 0.000 description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 6
- 244000061458 Solanum melongena Species 0.000 description 6
- 238000004925 denaturation Methods 0.000 description 6
- 230000036425 denaturation Effects 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- NDVLTYZPCACLMA-UHFFFAOYSA-N silver oxide Chemical compound [O-2].[Ag+].[Ag+] NDVLTYZPCACLMA-UHFFFAOYSA-N 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 241000607142 Salmonella Species 0.000 description 5
- 244000052616 bacterial pathogen Species 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- FJDQFPXHSGXQBY-UHFFFAOYSA-L caesium carbonate Chemical compound [Cs+].[Cs+].[O-]C([O-])=O FJDQFPXHSGXQBY-UHFFFAOYSA-L 0.000 description 5
- 229910000024 caesium carbonate Inorganic materials 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 241000588747 Klebsiella pneumoniae Species 0.000 description 4
- 108010079723 Shiga Toxin Proteins 0.000 description 4
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 4
- 229960000935 dehydrated alcohol Drugs 0.000 description 4
- 238000010511 deprotection reaction Methods 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 230000013595 glycosylation Effects 0.000 description 4
- 238000006206 glycosylation reaction Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 230000009545 invasion Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000012046 mixed solvent Substances 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 241000192125 Firmicutes Species 0.000 description 3
- 206010022653 Intestinal haemorrhages Diseases 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
- NWFNSTOSIVLCJA-UHFFFAOYSA-L copper;diacetate;hydrate Chemical compound O.[Cu+2].CC([O-])=O.CC([O-])=O NWFNSTOSIVLCJA-UHFFFAOYSA-L 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 239000000937 glycosyl acceptor Substances 0.000 description 3
- 239000005457 ice water Substances 0.000 description 3
- 210000000936 intestine Anatomy 0.000 description 3
- 150000004702 methyl esters Chemical class 0.000 description 3
- 229920000136 polysorbate Polymers 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 229910001923 silver oxide Inorganic materials 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000305071 Enterobacterales Species 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 241000588769 Proteus <enterobacteria> Species 0.000 description 2
- 244000297179 Syringa vulgaris Species 0.000 description 2
- 235000004338 Syringa vulgaris Nutrition 0.000 description 2
- 235000009754 Vitis X bourquina Nutrition 0.000 description 2
- 235000012333 Vitis X labruscana Nutrition 0.000 description 2
- 240000006365 Vitis vinifera Species 0.000 description 2
- 235000014787 Vitis vinifera Nutrition 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000003729 cation exchange resin Substances 0.000 description 2
- 229920001429 chelating resin Polymers 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000000348 glycosyl donor Substances 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- CASUWPDYGGAUQV-UHFFFAOYSA-M potassium;methanol;hydroxide Chemical compound [OH-].[K+].OC CASUWPDYGGAUQV-UHFFFAOYSA-M 0.000 description 2
- 230000005180 public health Effects 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 229920002379 silicone rubber Polymers 0.000 description 2
- 239000004945 silicone rubber Substances 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- UZASMYQTKALYAA-UHFFFAOYSA-N 1-benzothiophene 1H-pyrrole Chemical class N1C=CC=C1.S1C=CC2=C1C=CC=C2 UZASMYQTKALYAA-UHFFFAOYSA-N 0.000 description 1
- KVYRCBOUKXJXDK-UHFFFAOYSA-N 3,4-dimethylphenazine-1,2-diamine hydrochloride Chemical compound Cl.C1=CC=CC2=NC3=C(C)C(C)=C(N)C(N)=C3N=C21 KVYRCBOUKXJXDK-UHFFFAOYSA-N 0.000 description 1
- 240000001592 Amaranthus caudatus Species 0.000 description 1
- 235000009328 Amaranthus caudatus Nutrition 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 206010012741 Diarrhoea haemorrhagic Diseases 0.000 description 1
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Natural products CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 description 1
- 206010014896 Enterocolitis haemorrhagic Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000191070 Escherichia coli ATCC 8739 Species 0.000 description 1
- 208000032759 Hemolytic-Uremic Syndrome Diseases 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
- 102000005348 Neuraminidase Human genes 0.000 description 1
- 108010006232 Neuraminidase Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical class OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 241000588767 Proteus vulgaris Species 0.000 description 1
- 241001103617 Pseudomonas aeruginosa ATCC 15442 Species 0.000 description 1
- 125000003545 alkoxy group Chemical group 0.000 description 1
- 235000012735 amaranth Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000012300 argon atmosphere Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 101150118925 bioM gene Proteins 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 210000003555 cloaca Anatomy 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000000280 densification Methods 0.000 description 1
- 125000003963 dichloro group Chemical group Cl* 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000000147 enterotoxin Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 235000021050 feed intake Nutrition 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 125000005843 halogen group Chemical group 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 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
- 239000002808 molecular sieve Substances 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 229940007042 proteus vulgaris Drugs 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000007039 two-step reaction Methods 0.000 description 1
- 238000009777 vacuum freeze-drying 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
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/10—Enterobacteria
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Abstract
本发明公开了一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基。所述的显色培养基含有两种能产生不同颜色信号的显色底物和一种不显色但能产生非蓝色荧光信号的荧光底物;显色底物和荧光底物中除了含有β‑葡萄糖醛酸苷酶底物和β‑半乳糖苷酶底物外,还至少含有β‑葡萄糖苷酶底物、β‑N‑乙酰氨基葡萄糖苷酶底物、纤维二糖苷酶底物这三种所产颜色或荧光信号相同或相近底物中的一种底物。本发明提供的显色培养基对于更多的E.coli O157、non‑O157 E.coli以及非大肠杆菌,均能给出良好的检测特异性。
Description
技术领域
本发明涉及微生物检测技术领域,具体涉及一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基。
背景技术
大肠杆菌(Escherichia coli,E.coli)被普遍视为与食品、水、饲料、临床和环境样本卫生质量、清洁程度或致病风险密切相关的粪便污染指示菌。其中,E.coli O157可导致人类无症状的排泌、非血性腹泻、出血性结肠炎、溶血性尿毒综合症甚至死亡等不同后果的病症,是肠道出血性(或产志贺毒素)E.coli(EHEC或STEC)中最主要的致病血清型,已成为引起世界范围内广泛关注的一种重要致病菌。然而,除了E.coli O157之外,non-O157E.coli包括non-O157EHEC(或STEC)以及其它如肠道致病性E.coli(EPEC)、产肠毒素E.coli(ETEC)、肠道侵袭性E.coli(EIEC)、肠道集聚性E.coli(EAEC)以及其它仍需进一步流行病学研究的致病型如弥散粘附性E.coli(DAEC)等也能导致人类腹泻等病症爆发,也是需要引起重视的(Gomes et al.,Brazilian Journal of Microbiology,2016,47:3-30;Mead et al.,Lancet,1998,352:1207-1212;Pennington et al.,Lancet,2010,376:1428-1435;Yang et al.,Archives of Microbiology,2017,199:811-825.)。因此,检测E.coliO157与non-O157E.coli,对于保障食品安全等公共卫生安全是必不可少的。
尽管目前已有多种关于检测E.coli的显色培养基被发展出来,但是它们仍然难以满足这样的“一款多用”目的,即在相对较高特异地检测E.coli O157与non-O157E.coli上均可应用。例如,TBX琼脂(Tryptone Bile X-glucuronide agar)已被国际标准(ISO16649-3:2015和ISO/TS 13136:2012)推荐应用于计数具有β-葡萄糖醛酸苷酶(β-glucuronidase)活性的E.coli与分离包括无β-葡萄糖醛酸苷酶活性E.coli O157在内等STEC,但它只含有X-glucuronide一种显色底物,只能使具有β-葡萄糖醛酸苷酶活性的non-O157E.coli显蓝绿色,而不具有β-葡萄糖醛酸苷酶活性的E.coli O157与很多种杂菌均显无色,导致E.coli O157难以被辨别,使检测、鉴定工作量大幅度增加。Chromocult(Merck,德国)、Coli ID(bioMérieux,法国)、CHROMagar ECC(Chromagar,法国)、HiCrome ECC(Union Carbide,美国)等这些显色培养基添加了两种不同酶(β-半乳糖苷酶与β-葡萄糖醛酸苷酶)显色底物,可用于检测大肠菌群和具有β-葡萄糖醛酸苷酶活性的non-O157E.coli,但是因与其它大肠菌群都显相同的颜色而难以鉴别出E.coli O157(Manafi M.,International Journal of Food Microbiology,2000,60:205-218.)。而E.coli O157显色培养基又只适用于特异性检测E.coli O157,对non-O157E.coli的检测效果比较差,例如CHROMagar O157(Chromagar,法国)、Rainbow Agar O157(Biolog Inc.,美国)和RapidE.coli O157:H7(Bio-Rad)等等(Verhaegen et al.,International Journal ofEnvironmental Research and Public Health,2015,12:6965-6978;Fan et al.,International Journal of Food Microbiology,2018,266:295-300.)。
发明内容
本发明的目的在于提供一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基。
本发明通过以下技术方案来实现:
一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基,不含有山梨醇、也不含有中性红,但含有两种能产生无扩散性的不同颜色信号的显色底物和一种不显色但能产生无扩散性的非蓝色荧光信号的荧光底物,所述的显色底物和荧光底物中除了含有β-葡萄糖醛酸苷酶底物和β-半乳糖苷酶底物外,还至少含有β-葡萄糖苷酶底物、β-N-乙酰氨基葡萄糖苷酶底物、纤维二糖苷酶底物这三种所产颜色或荧光信号相同或相近底物中的一种底物。
所述的显色底物为基于3-吲哚酚衍生物的吲哚酚基显色底物,所述的3-吲哚酚衍生物的结构如下式(I)所示:
上式中的R可以为单取代基团,也可为多取代基团。
优选,所述的3-吲哚酚衍生物为5-溴-4-氯-3-吲哚酚、5-溴-6-氯-3-吲哚酚或6-氯-3-吲哚酚等。
优选,所述的基于3-吲哚酚衍生物的吲哚酚基显色底物为5-溴-4-氯-3-吲哚基-β-D-葡萄糖苷、5-溴-6-氯-3-吲哚基-β-D-半乳糖苷、5-溴-4-氯-3-吲哚基-β-D-葡萄糖醛酸苷环己胺盐或5-溴-6-氯-3-吲哚基-β-D-葡萄糖醛酸苷环己胺盐。
所述的荧光底物为基于2-(苯并噻唑-2'-基)苯酚衍生物的的荧光底物,所述的2-(苯并噻唑-2'-基)苯酚衍生物的结构如下式(2)所示:
上式中的R1和R2代表卤原子或烷氧基等基团。
优选,所述的2-(苯并噻唑-2'-基)苯酚衍生物为2-(苯并噻唑-2'-基)-4-溴苯酚等。
优选,所述的基于2-(苯并噻唑-2'-基)苯酚衍生物的的荧光底物为2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖醛酸苷、2-(苯并噻唑-2'-基)-4-溴苯基-β-D-半乳糖苷或2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖苷。
所述的荧光物2-(苯并噻唑-2'-基)苯酚衍生物是根据文献[Otsubo etal.Bioorganic&
Medicinal Chemistry Letters,2013,23(7):2245-2249]报道的方法合成。而基于2-(苯并噻唑-2'-基)苯酚衍生物的荧光底物的合成方法包括两步反应,具体步骤为:
S1:以2-(苯并噻唑-2'-基)苯酚衍生物为糖基受体,相应的乙酰卤代糖为糖基供体,氧化银或碳酸铯为催化剂,乙腈为溶剂,进行糖基化反应,得到含保护基团乙酰基的目标糖苷底物;
S2:将得到的含保护基团乙酰基的目标糖苷底物置于含氢氧化钾或氢氧化锂的甲醇和四氢呋喃混合体系中进行脱保护基反应,得到目标荧光底物。
上述合成方法中,对于相应的β-葡萄糖醛酸苷合成时,优选氢氧化锂作为碱;而对于其它糖苷合成时,优选氢氧化钾作为碱。
所述的催化剂(氧化银或碳酸铯)与糖基供体(乙酰卤代糖)在投料摩尔量上均多于糖基受体(2-(苯并噻唑-2'-基)苯酚衍生物),优选,所述的催化剂:糖基供体:糖基受体的投料摩尔比为1.5:1.5:1.0。
本发明用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基,还含有十二烷基硫酸钠(SDS)和(或)胆盐(3号胆盐、脱氧胆酸钠等)、细菌存活和生长所需的成分(如蛋白胨、酵母提取粉、氯化钠等)、琼脂、异丙基-β-D-硫代吡喃半乳糖苷(IPTG)以及可能含有的少量乳糖、水杨苷或纤维二糖、吐温80或曲拉通X-100。
优选,所述的显色培养基中显色底物和荧光底物的浓度均为0.01~0.30g/L。
优选,所述的显色培养基还含有十二烷基硫酸钠0.05~0.25g/L和/或胆盐0~1.50g/L、蛋白胨1~20g/L、酵母提取粉0~6g/L、氯化钠4~8g/L、琼脂10~20g/L、异丙基-β-D-硫代吡喃半乳糖苷0.01~0.10g/L。
优选,所述的显色培养基还含有乳糖、水杨苷或纤维二糖0.01~0.15g/L。
优选,所述的显色培养基还含有吐温80 0~0.60mL/L或曲拉通X-100 0~0.20mL/L。
所述的β-葡萄糖醛酸苷酶底物为羧酸形式或羧酸盐形式。
与现有技术相比,本发明的优势在于:
本发明用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基可以在显色培养基种类选择以及相关处理工作(如显色培养基使用前的平板制备工作等)上投入减少的情况下,仍然可以满足既能用于检测E.coli O157、也能用于检测非O157E.coli的多重目的,并且检测的特异性相对较高。
附图说明
图1为荧光底物2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖醛酸苷、2-(苯并噻唑-2'-基)-4-溴苯基-β-D-半乳糖苷或2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖苷的合成路线。
图2为实施例4的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基对E.coli O157ATCC 35150、E.coli ATCC 25922、阪崎肠杆菌ATCC 29544和鼠伤寒沙门氏菌ATCC14028四种不同致病菌在37℃下有氧培养18h后的检测效果。其中,平板中右上方、左上方、左下方、右下方依次为E.coli O157ATCC 35150、E.coli ATCC 25922、阪崎肠杆菌ATCC29544和鼠伤寒沙门氏菌ATCC 14028,A为可见光下,B为365nm紫外光下,C为可见光和手电筒375nm紫外光下。
图3为实施例4、5、6的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基对E.coli O157、non-O157E.coli以及非大肠杆菌在37℃下分别有氧培养12h、18h后的检测效果。其中,A:实施例4组,B:实施例5组,C:实施例6组;下标1表示在可见光下观察,下标2表示在365nm紫外灯光下观察,Ⅰ为A至C显色培养基平板中各菌对应的编号。
图4为实施例7的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基对E.coli O157、non-O157E.coli以及非大肠杆菌在37℃下分别有氧培养12h、18h后的检测效果。其中,左图表示在可见光下观察,右图表示在365nm紫外灯光下观察;Ⅰ为显色培养基平板中各菌对应的编号。
图5为一些已有培养基对E.coli O157、non-O157E.coli以及非大肠杆菌在37℃下分别有氧培养12h、18h后的检测效果。其中,D:TBX琼脂组,E:CHROMagar E.coli组,F:CHROMagar O157组;G:山梨醇-麦康凯培养基(SMAC)组,下标1表示在可见光下观察,下标2表示在365nm紫外灯光下观察;Ⅰ为D至G显色培养基平板中各菌对应的编号。
具体实施方式
以下实施例是对本发明的进一步说明,而不是对本发明的限制。
实施例1 2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖醛酸苷的合成
(1)糖基化反应
将0.612g(2.00mmol)荧光物2-(苯并噻唑-2'-基)-4-溴苯酚(1)、0.695g(3.00mmol,1.5e.q.)氧化银、1.192g(3.00mmol,1.5e.q.)三乙酰基-α-D-溴代葡萄糖醛酸甲酯(2a)置于含2.0g 4A分子筛的16mL干燥乙腈中,并于氩气氛围、避光和室温条件下搅拌反应32h。然后,将反应混合物用装填有致密的300-400目柱层层析硅胶层的砂芯漏斗抽滤,用二氯甲烷和乙酸乙酯混合液(v/v=2/1)洗涤,合并洗脱液并减压旋蒸去除溶剂,依次用无水乙醇、乙酸乙酯重结晶,抽滤,干燥后可得约0.348g(产率约28%)含保护基的目标糖苷底物(3a)。
或者,将0.306g(1.00mmol)荧光物2-(苯并噻唑-2'-基)-4-溴苯酚(1)、0.652g(2.00mmol)碳酸铯置于10mL干燥乙腈中搅拌10min后,加入0.596g(1.50mmol)三乙酰基-α-D-溴代葡萄糖醛酸甲酯(2a),室温下搅拌反应2h。然后,将反应混合物用装填有致密的300-400目柱层层析硅胶层的砂芯漏斗抽滤,用二氯甲烷和乙酸乙酯混合液(v/v=2/1)洗涤,合并洗脱液并减压旋蒸去除溶剂,依次用无水乙醇、乙酸乙酯重结晶,抽滤,干燥可得到约0.137g(产率约22%)含保护基的目标糖苷底物(结构式见式3a)。
1H-NMR(500MHz,CDCl3):δ=8.65(s,1H),8.10(d,J=8.0Hz,1H),7.94(d,J=8.0Hz,1H),7.55(d,J=8.7Hz,1H),7.51(t,J=7.6Hz,1H),7.41(t,J=7.5Hz,1H),7.11(d,J=8.8Hz,1H),5.52(t,J=8.0Hz,1H),5.45(t,J=9.3Hz,1H),5.39(d,J=7.5Hz,1H),5.35(t,J=9.3Hz,1H),4.27(d,J=9.4Hz,1H),3.71(s,3H),2.06(s,3H),2.05(s,3H),1.81(s,3H)ppm.13C-NMR(126MHz,CDCl3):δ=170.12,169.29,166.59,160.77,153.03,151.80,136.10,134.35,132.49,126.37,125.41,125.19,123.14,121.39,116.95,116.55,98.97,72.85,72.08,71.10,68.70,53.11,20.61,20.55,20.52ppm.HRMS(ESI):m/z[M+H]+Calcd.for C26H25BrNO10S:622.0377;found:622.0375。
(2)脱保护基反应
取0.200g(0.32mmol)上述步骤(1)所得含保护基的目标糖苷底物(3a)置于12mL无水的甲醇和四氢呋喃混合溶剂(v/v=1/2)中,冰水浴冷却下加入8mg(0.32mmol,1.0e.q.)LiOH,于2~8℃下搅拌反应6h,然后加入Amberlite IR-120(H+)氢型阳离子交换树脂中和,过滤,将滤液于40±2℃水浴下减压旋蒸去除溶剂后,加入12mL甲醇和四氢呋喃混合溶剂(v/v=1/1),冰水浴冷却后,加入0.96mL LiOH(0.5mol/L)水溶液,于冰水浴下搅拌反应6h,然后加入6mL四氢呋喃,并加入Amberlite IR-120(H+)氢型阳离子交换树脂调pH值至4~5,过滤,将滤液置于30~35℃水浴下减压旋蒸去除有机溶剂,再真空冷冻干燥,然后加入二氯甲烷充分搅拌,过滤,再将固体置于二氯甲烷中充分搅拌,过滤,重复3次后,用乙腈洗涤,干燥后得到约79mg(产率约51%)最终目标产物2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖醛酸苷(结构式见式4a)。
1H-NMR(500MHz,DMSO-d6):δ=8.55(s,1H),8.13(d,J=8.1Hz,1H),8.10(d,J=8.0Hz,1H),7.73(d,J=8.4Hz,1H),7.57(t,J=7.2Hz,1H),7.48(t,J=6.7Hz,1H),7.38(d,J=8.5Hz,1H),5.55(d,J=4.5Hz,1H),5.46(d,J=7.5Hz,1H),5.39(s,1H),4.03(d,J=9.5Hz,1H),3.65–3.68(m,1H),3.45(t,J=9.3Hz,1H),3.42–3.35(m,2H)ppm.13C-NMR(126MHz,DMSO-d6):δ=169.98,160.68,153.49,151.34,135.92,134.39,130.63,126.45,125.38,123.69,122.76,121.87,117.22,113.94,99.77,76.02,75.45,72.89,71.25ppm.HRMS(ESI):m/z[M+H]+Calcd.for C19H17BrNO7S:481.9904;found:481.9906。
上述实施例的目标荧光底物2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖醛酸苷(4a)的合成路线见图1。
实施例2 2-(苯并噻唑-2'-基)-4-溴苯基-β-D-半乳糖苷的合成
2-(苯并噻唑-2'-基)-4-溴苯基-β-D-半乳糖苷(4b)的合成路线见图1,具体步骤如下:
(1)糖基化反应
将0.306g(1.00mmol)荧光物2-(苯并噻唑-2′-基)-4-溴苯酚(1)、0.652g(2.00mmol)碳酸铯置于10mL干燥乙腈中搅拌10min后,加入0.617g(1.50mmol)四乙酰基-α-D-溴代半乳糖(2b),室温下搅拌反应2h。过滤,二氯甲烷洗涤滤渣,合并滤液,将滤液置于40~45℃水浴下加压旋蒸去除溶剂,用无水乙醇重结晶,干燥可得到约0.600g(产率约94%)含保护基的目标糖苷底物(结构式见式3b)。
1H-NMR(500MHz,CDCl3):δ=8.69(d,J=2.5Hz,1H),8.13(d,J=8.0Hz,1H),7.94(d,J=8.0Hz,1H),7.55(dd,J=8.6,2.3Hz,1H),7.52(d,J=7.5Hz,1H),7.42(t,J=7.5Hz,1H),7.12(d,J=8.8Hz,1H),5.72(dd,J=10.0,8.5Hz,1H),5.49(d,J=3.0Hz,1H),5.30(d,J=8.0Hz,1H),5.14(dd,J=10.2,3.2Hz,1H),4.28–4.21(m,1H),4.20–4.13(m,2H),2.22(s,3H),2.08(s,3H),2.02(s,3H),1.81(s,3H)ppm.13C NMR(126MHz,CDCl3):δ=170.43,170.33,170.23,169.43,161.11,153.31,151.74,136.19,134.41,132.64,126.56,125.56,125.10,123.22,121.48,116.88,116.45,99.38,71.66,71.41,68.69,66.92,61.51,20.86,20.81,20.70ppm。
(2)脱保护基反应
将0.500g(0.78mmol)上述步骤(1)所得含保护基的目标糖苷底物(3b)置于15mL无水的甲醇/四氢呋喃混合溶剂(v/v=1/1)中,加入11.8μL氢氧化钾甲醇溶液(1.0mol/L;0.15e.q.),室温下搅拌反应1h。然后于40~45℃水浴中旋蒸去除溶剂,然后加入二氯甲烷搅拌,抽滤,用二氯甲烷洗涤多次,接着用冷水洗涤2次,最后用少量乙腈洗涤2次,干燥后可得到约0.364g(产率约99%)最终目标目标产物2-(苯并噻唑-2'-基)-4-溴苯基-β-D-半乳糖苷(结构式见式4b)。
1H-NMR(500MHz,DMSO-d6):δ=8.55(s,1H),8.12(d,J=8.0Hz,1H),8.10(d,J=8.5Hz,1H),7.70(dd,J=9.0,2.0Hz,1H),7.56(t,J=7.5Hz,1H),7.47(t,J=7.3Hz,1H),7.36(d,J=9.0Hz,1H),5.23(s,1H),5.22(s,1H),5.01(d,J=5.5Hz,1H),4.70(t,J=5.2Hz,1H),4.66(d,J=4.0Hz,1H),3.94(dd,J=14.1,8.1Hz,1H),3.76(s,1H),3.71(t,J=5.5Hz,1H),3.61–3.47(m,3H)ppm.13C-NMR(126MHz,DMSO):δ=160.85,153.98,151.34,135.98,134.30,130.46,126.38,125.28,123.54,122.69,121.81,117.42,113.52,100.96,75.82,73.51,70.10,68.02,60.30ppm。
实施例3 2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖苷的合成
2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖苷(4c)的合成路线见图1,具体步骤如下:
(1)糖基化反应
将0.306g(1.00mmol)荧光物2-(苯并噻唑-2′-基)-4-溴苯酚(1)、0.652g(2.00mmol)碳酸铯置于10mL干燥乙腈中搅拌10min后,加入0.617g(1.50mmol)四乙酰基-α-D-溴代葡萄糖(2c),室温下搅拌反应2h。过滤,二氯甲烷洗涤滤渣,合并滤液,将滤液置于40~45℃水浴下加压旋蒸去除溶剂,用无水乙醇重结晶,干燥可得到约0.523g(产率约82%)含保护基的目标糖苷底物(结构式见式3c)。
1H-NMR(500MHz,DMSO-d6):δ=8.52(d,J=2.5Hz,1H),8.11(d,J=8.0Hz,1H),8.05(d,J=8.0Hz,1H),7.81(dd,J=9.0,2.5Hz,1H),7.60–7.56(m,1H),7.51–7.48(m,1H),7.35(d,J=9.0Hz,1H),5.99(d,J=8.0Hz,1H),5.43(t,J=9.5Hz,1H),5.36(dd,J=9.5,8.0Hz,1H),5.10(t,J=9.5Hz,1H),4.35(ddd,J=10.0,5.5,2.5Hz,1H),4.21(dd,J=12.5,5.5Hz,1H),4.11(dd,J=12.4,2.3Hz,1H),2.03,1.99,1.98,1.88(4×s,4×3H)ppm.13C-NMR(126MHz,DMSO-d6):δ=170.42,170.14,169.79,169.54,160.44,153.20,151.80,135.97,135.05,131.51,127.13,126.20,124.41,123.43,122.02,118.17,115.43,97.18,72.87,71.64,71.20,68.11,61.94,20.93,20.88,20.85,20.76ppm.
(2)脱保护基反应
将0.300g(0.47mmol)上述步骤(1)所得含保护基的目标糖苷底物(3c)置于9mL无水的甲醇/四氢呋喃混合溶剂(v/v=1/1)中,加入7.0μL氢氧化钾甲醇溶液(1.0mol/L;0.15e.q.),室温下搅拌反应1h。然后于40~45℃水浴中旋蒸去除溶剂,然后加入二氯甲烷搅拌,抽滤,用二氯甲烷洗涤多次,接着用冷水洗涤2次,最后用少量乙腈洗涤2次,干燥后可得到约0.219g(产率约99%)最终目标目标产物2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖苷(结构式见式4c)。
1H-NMR(500MHz,DMSO-d6):δ=8.55(d,J=2.5Hz,1H),8.11(dd,J=12.3,8.0Hz,2H),7.70(dd,J=9.0,3.0Hz,1H),7.58–7.54(m,1H),7.49–7.45(m,1H),7.36(d,J=9.5Hz,1H),5.42(d,J=5.5Hz,1H),5.29(d,J1,2=7.5Hz,1H),5.26(s,1H),5.13(d,J=3.5Hz,1H),4.62(t,J=5.3Hz,1H),3.71(dd,J=10.5,5.5Hz,1H),3.62(dd,J=13.7,8.2Hz,1H),3.53–3.45(m,2H),3.37(d,J=9.5Hz,1H),3.26(dt,J=13.2,6.7Hz,1H)ppm.13C-NMR(126MHz,DMSO-d6):δ=161.31,154.34,151.81,136.44,134.79,130.95,126.86,125.76,124.04,123.17,122.32,117.88,114.10,100.71,77.72,77.31,73.63,69.98,61.01ppm.
实施例4
一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基,其每1000mL含有10g蛋白胨、3g酵母提取粉、5g氯化钠、0.1g SDS、0.1g脱氧胆酸钠、0.5mL吐温80、0.15g荧光底物2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖醛酸苷(于365~375nm紫外光下能产黄绿色荧光)、0.05g显色底物5-溴-4-氯-3-吲哚基-β-D-葡萄糖苷(能产蓝至蓝绿色)、0.05g显色底物5-溴-6-氯-3-吲哚基-β-D-半乳糖苷(能产紫红色)、0.05g IPTG、15g琼脂。(注:该显色培养基可以于121℃下高温蒸汽灭菌15min)
实施例5
一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基,其每1000mL含有10g蛋白胨、3g酵母提取粉、5g氯化钠、0.1g SDS、0.1g脱氧胆酸钠、0.045g荧光底物2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖苷(于365~375nm紫外光下能产黄绿色荧光)、0.20g显色底物5-溴-4-氯-3-吲哚基-β-D-葡萄糖醛酸苷环己胺盐(能产蓝至蓝绿色)、0.05g显色底物5-溴-6-氯-3-吲哚基-β-D-半乳糖苷(能产紫红色)、0.05g IPTG、0.01g水杨苷、0.01g纤维二糖、15g琼脂。(注:该显色培养基也可以于121℃下高温蒸汽灭菌15min)
实施例6
一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基,其每1000mL含有10g蛋白胨、3g酵母提取粉、5g氯化钠、0.1g SDS、0.1g脱氧胆酸钠、0.5mL吐温80、0.045g荧光底物2-(苯并噻唑-2′-基)-4-溴苯基-β-D-半乳糖苷(于365~375nm紫外光下能产黄绿色荧光)、0.20g显色底物5-溴-6-氯-3-吲哚基-β-D-葡萄糖醛酸苷环己胺盐(能产紫红色)、0.055g显色底物5-溴-4-氯-3-吲哚基-β-D-葡萄糖苷(能产蓝至蓝绿色)、0.025g IPTG、0.025g乳糖、15g琼脂。(注:该显色培养基也可以于121℃下高温蒸汽灭菌15min)
实施例7
一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基,其每1000mL含有10g蛋白胨、3g酵母提取粉、5g氯化钠、0.20g SDS、0.20g脱氧胆酸钠、0.10mL曲拉通X-100、0.15g荧光底物2-(苯并噻唑-2′-基)-4-溴苯基-β-D-葡萄糖醛酸苷(于365~375nm紫外光下能产黄绿色荧光)、0.05g显色底物5-溴-4-氯-3-吲哚基-β-D-葡萄糖苷(能产蓝至蓝绿色)、0.05g显色底物5-溴-6-氯-3-吲哚基-β-D-半乳糖苷(能产紫红色)、0.05g IPTG、琼脂15g。(注:该显色培养基可以于121℃下高温蒸汽灭菌15min)
实施例8
一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基,其每1000mL含有1g蛋白胨、6g酵母提取粉、4g氯化钠、0.05g SDS、1.5g 3号胆盐、0.20mL曲拉通X-100、0.05g荧光底物2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖苷(于365~375nm紫外光下能产黄绿色荧光)、0.20g显色底物5-溴-4-氯-3-吲哚基-β-D-葡萄糖醛酸苷环己胺盐(能产蓝至蓝绿色)、0.05g显色底物5-溴-6-氯-3-吲哚基-β-D-半乳糖苷(能产紫红色)、0.1g IPTG、0.15g水杨苷、10g琼脂。(注:该显色培养基也可以于121℃下高温蒸汽灭菌15min)
实施例9
一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基,其每1000mL含有20g蛋白胨、8g氯化钠、0.25g SDS、0.60mL吐温80、0.05g荧光底物2-(苯并噻唑-2′-基)-4-溴苯基-β-D-半乳糖苷(于365~375nm紫外光下能产黄绿色荧光)、0.30g显色底物5-溴-6-氯-3-吲哚基-β-D-葡萄糖醛酸苷环己胺盐(能产紫红色)、0.01g显色底物5-溴-4-氯-3-吲哚基-β-D-葡萄糖苷(能产蓝至蓝绿色)、0.05g显色底物5-溴-4-氯-3-吲哚基-β-D-N-乙酰氨基葡萄糖苷(能产蓝至蓝绿色)、0.20g显色底物5-溴-4-氯-3-吲哚基-β-D-纤维二糖苷(能产蓝至蓝绿色)、0.15g乳糖、0.15g纤维二塘、20g琼脂。(注:该显色培养基也可以于121℃下高温蒸汽灭菌15min)
实施例10
评价本发明实施例4的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基对E.coli O157:H7ATCC 35150、E.coli ATCC 25922、阪崎肠杆菌ATCC 29544和鼠伤寒沙门氏菌ATCC 14028这四种不同致病菌的检测效果,使用1μL蓝色接种环蘸取相应的纯菌培养液并划线接种于显色培养基中,于37℃下有氧培养18h。然后分别在可见光、365nm紫外光、可见光和手电筒375nm紫外光下观察显色培养基对四种不同致病菌的区分度,结果见图2。
图2平板中右上方、左上方、左下方、右下方依次为E.coli O157:H7ATCC 35150、E.coli ATCC 25922、阪崎肠杆菌ATCC 29544和鼠伤寒沙门氏菌ATCC 14028,A为可见光下,B为365nm紫外光下,C为可见光和手电筒375nm紫外光下。如图2所示,实施例4的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基对以下这四种致病菌能给出良好的区分度:E.coli O157:H7ATCC 35150(右上方)显粉色且无荧光,E.coli ATCC 25922(左上方)显粉色且有黄绿色荧光,阪崎肠杆菌ATCC 29544(左下方)显蓝绿色且无荧光,鼠伤寒沙门氏菌ATCC 14028(右下方)显无色且无荧光。
实施例11
分别评价本发明实施例4、5、6的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基对E.coli O157、non-O157E.coli以及非大肠杆菌的检测效果。接种方法为使用2.5μL移液枪吸取相应的纯菌培养液并通过反向移液法小心点种0.2μL至平板上相应的位置;于37℃下有氧培养12h、18h。然后分别在可见光、365nm紫外灯光下观察。结果见图3。
图3中,A:实施例4组,B:实施例5组,C:实施例6组;下标1表示在可见光下观察,下标2表示在365nm紫外灯光下观察;Ⅰ为A至C显色培养基平板中各菌对应的编号,实验菌株有:①肠道集聚性E.coli(EAEC)分离株(No.1288-1),②EAEC分离株(No.3476B3),③AEC分离株(No.3164A1),④肠道侵袭性E.coli(EIEC)分离株(No.2968A1),⑤EIEC分离株(No.2987A2),⑥肠道致病性E.coli(EPEC)分离株(No.2684-2),⑦EPEC分离株(No.2627-2),⑧EPEC分离株(No.545-1),⑨产志贺毒素(或肠道出血性)E.coli(STEC或EHEC)分离株(No.T14259B3),⑩0STEC或EHEC分离株(No.T53424A1),STEC或EHEC分离株(No.T74274A1),STEC或EHEC分离株(No.T183724C1),STEC或EHEC分离株(No.T911964-1),STEC或EHEC分离株(No.T1111122-3),E.coli O157:H7ATCC 35150,E.coli O157:H7NCTC 12900,E.coliO157:H7882364,E.coli O157:H7ATCC 43895,E.coli O157:H7ATCC 43888,E.coli CMCC(B)44102,E.coli CMCC(B)44103,E.coli ATCC 8739,E.coli ATCC25922,E.coli ATCC 8099,E.coli ATCC 35218,E.coli CMCC(B)44113,E.coliHK 70115,产气肠杆菌CMCC(B)45103,阴沟肠杆菌CMCC(B)45301,肺炎克雷伯氏菌CMCC(B)46117,肺炎克雷伯氏菌广临检-57,阪崎肠杆菌ATCC 29544,阪崎肠杆菌分离株(No.10Ta),阪崎肠杆菌分离株(No.2864C1),阪崎肠杆菌分离株(No.1541A1-1),鼠伤寒沙门氏菌ATCC 14028,鼠伤寒沙门氏菌分离株(No.81-8),鼠伤寒沙门氏菌分离株(No.97-1),肠炎沙门氏菌CMCC(B)50335,伤寒沙门氏菌CMCC(B)50071,普通变性杆菌CMCC(B)49027,奇异变性杆菌CMCC(B)49005,铜绿假单胞菌CMCC(B)10104,铜绿假单胞菌ATCC 15442,铜绿假单胞菌ATCC27853,铜绿假单胞菌ATCC 9027,铜绿假单胞菌GIM 1.46,蜡样芽胞杆菌ATCC14579。
如图3所示,在实施例4(A)中,E.coli O157:H7全部显粉色且无荧光,non-O157E.coli全部显粉色且有黄绿色荧光,non-E.coli显绿色(见阪崎肠杆菌)、蓝紫色(见产气肠杆菌、阴沟肠杆菌、1株肺炎克雷伯菌)、金属蓝色(见1株肺炎克雷伯菌)、或不显色(见鼠伤寒沙门氏菌、肠炎沙门氏菌、伤寒沙门氏菌、铜绿假单胞菌、变形杆菌),革兰氏阳性菌受抑制(见蜡样芽胞杆菌ATCC 14579)。
在实施例5(B)中,E.coli O157:H7全部显粉色且无荧光,non-O157E.coli全部显金属蓝色且无荧光,non-E.coli显粉色或淡紫红色且有黄绿色荧光(见产气肠杆菌、阴沟肠杆菌和肺炎克雷伯菌)、无色且无荧光(见沙门氏菌、变形杆菌和部分铜绿假单胞菌)、或无色且有蓝色荧光(见部分铜绿假单胞菌),革兰氏阳性菌受抑制(见蜡样芽胞杆菌ATCC14579)。
在实施例6(C)中,E.coli O157:H7全部显无色且有米黄色荧光,non-O157E.coli全部显紫红色且无明显荧光,non-E.coli显绿色或蓝绿色(见阪崎肠杆菌、产气肠杆菌、阴沟肠杆菌、肺炎克雷伯菌)、不显色且无荧光或有蓝色荧光(见部分铜绿假单胞菌、变形杆菌),革兰氏阳性菌受抑制(见蜡样芽胞杆菌ATCC 14579)。
由此可见,对于更多的E.coli O157、non-O157E.coli以及非大肠杆菌,实施例4、5和6的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基也都能给出良好的检测特异性。
实施例12
评价本发明实施例7的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基对E.coli O157、non-O157E.coli以及非大肠杆菌的检测效果。接种方法为使用2.5μL移液枪吸取相应的纯菌培养液并通过反向移液法小心点种0.2μL至平板上相应的位置;于37℃下有氧培养12h、18h。然后分别在可见光、365nm紫外灯光下观察。结果见图4。
图4中,左图表示在可见光下观察,右图表示在365nm紫外灯光下观察;Ⅰ为显色培养基平板中各菌对应的编号,实验菌株有:①肠道集聚性E.coli(EAEC)分离株(No.1288-1),②EAEC分离株(No.3476B3),③AEC分离株(No.3164A1),④肠道侵袭性E.coli(EIEC)分离株(No.2968A1),⑤EIEC分离株(No.2987A2),⑥肠道致病性E.coli(EPEC)分离株(No.2684-2),⑦EPEC分离株(No.2627-2),⑧EPEC分离株(No.545-1),⑨产志贺毒素(或肠道出血性)E.coli(STEC或EHEC)分离株(No.T14259B3),⑩0STEC或EHEC分离株(No.T53424A1),STEC或EHEC分离株(No.T74274A1),STEC或EHEC分离株(No.T183724C1),STEC或EHEC分离株(No.T911964-1),STEC或EHEC分离株(No.T1111122-3),E.coli O157:H7ATCC 35150,E.coli O157:H7NCTC 12900,E.coliO157:H7882364,E.coli O157:H7ATCC 43895,E.coliO157:H7ATCC 43888,E.coliCMCC(B)44102,E.coli CMCC(B)44103,E.coliATCC 8739,E.coli ATCC 25922,E.coli ATCC 8099,E.coli ATCC 35218,E.coli CMCC(B)44113,E.coli HK 70115,产气肠杆菌CMCC(B)45103,阴沟肠杆菌CMCC(B)45301,肺炎克雷伯氏菌CMCC(B)46117,肺炎克雷伯氏菌广临检-57,阪崎肠杆菌ATCC 29544,阪崎肠杆菌分离株(No.10Ta),阪崎肠杆菌分离株(No.2864C1),阪崎肠杆菌分离株(No.1541A1-1),鼠伤寒沙门氏菌ATCC 14028,鼠伤寒沙门氏菌分离株(No.81-8),鼠伤寒沙门氏菌分离株(No.97-1),肠炎沙门氏菌CMCC(B)50335,伤寒沙门氏菌CMCC(B)50071,普通变性杆菌CMCC(B)49027,奇异变性杆菌CMCC(B)49005,铜绿假单胞菌CMCC(B)10104,铜绿假单胞菌ATCC15442,铜绿假单胞菌ATCC 27853,铜绿假单胞菌ATCC 9027,铜绿假单胞菌GIM1.46,蜡样芽胞杆菌ATCC 14579。
如图3和图4所示,对比实施例4和实施例7可知,通过适当地控制SDS和胆盐的用量等措施,可更好地抑制背景菌的生长,从而提高对O157/non-O157E.coli的选择性。
实施例13
分别评价TBX琼脂、CHROMagar E.coli、CHROMagar O157、山梨醇-麦康凯培养基这四种现有的市售培养基对E.coli O157、non-O157E.coli以及非大肠杆菌的检测效果。接种方法为使用2.5μL移液枪吸取相应的纯菌培养液并通过反向移液法小心点种0.2μL至平板上相应的位置;于37℃下有氧培养12h、18h。然后分别在可见光、365nm紫外灯光下观察。结果见图5。
图5中,D:TBX琼脂组,E:CHROMagar E.coli组,F:CHROMagar O157组;G:山梨醇-麦康凯培养基(SMAC)组,下标1表示在可见光下观察,下标2表示在365nm紫外灯光下观察;Ⅰ为D至G显色培养基平板中各菌对应的编号,实验菌株有:①肠道集聚性E.coli(EAEC)分离株(No.1288-1),②EAEC分离株(No.3476B3),③AEC分离株(No.3164A1),④肠道侵袭性E.coli(EIEC)分离株(No.2968A1),⑤EIEC分离株(No.2987A2),⑥肠道致病性E.coli(EPEC)分离株(No.2684-2),⑦EPEC分离株(No.2627-2),⑧EPEC分离株(No.545-1),⑨产志贺毒素(或肠道出血性)E.coli(STEC或EHEC)分离株(No.T14259B3),⑩STEC或EHEC分离株(No.T53424A1),STEC或EHEC分离株(No.T74274A1),STEC或EHEC分离株(No.T183724C1),STEC或EHEC分离株(No.T911964-1),STEC或EHEC分离株(No.T1111122-3),E.coli O157:H7ATCC35150,E.coli O157:H7NCTC 12900,E.coliO157:H7882364,E.coli O157:H7ATCC 43895,E.coli O157:H7ATCC 43888,E.coliCMCC(B)44102,E.coliCMCC(B)44103,E.coli ATCC 8739,E.coli ATCC 25922,E.coli ATCC 8099,E.coli ATCC 35218,E.coli CMCC(B)44113,E.coli HK 70115,产气肠杆菌CMCC(B)45103,阴沟肠杆菌CMCC(B)45301,肺炎克雷伯氏菌CMCC(B)46117,肺炎克雷伯氏菌广临检-57,阪崎肠杆菌ATCC 29544,阪崎肠杆菌分离株(No.10Ta),阪崎肠杆菌分离株(No.2864C1),阪崎肠杆菌分离株(No.1541A1-1),鼠伤寒沙门氏菌ATCC 14028,鼠伤寒沙门氏菌分离株(No.81-8),鼠伤寒沙门氏菌分离株(No.97-1),肠炎沙门氏菌CMCC(B)50335,伤寒沙门氏菌CMCC(B)50071,普通变性杆菌CMCC(B)49027,奇异变性杆菌CMCC(B)49005,铜绿假单胞菌CMCC(B)10104,铜绿假单胞菌ATCC 15442,铜绿假单胞菌ATCC 27853,铜绿假单胞菌ATCC 9027,铜绿假单胞菌GIM 1.46,蜡样芽胞杆菌ATCC 14579。
如图5所示,在TBX琼脂和CHROMagar E.coli上,除了E.coli ATCC 8739不显色或蓝绿色较淡外,其余non-O157E.coli均呈现明显的蓝绿色,而E.coli O157:H7与全部non-E.coli菌株均显无色而难以区分。在CHROMagar O157上,E.coli O157:H7显淡紫红色,鼠伤寒沙门氏菌、伤寒沙门氏菌显粉色,而其余non-O157E.coli与产气肠杆菌、阴沟肠杆菌、肺炎克雷伯氏菌以及阪崎肠杆菌均显金属蓝或蓝绿色而难以区分。在山梨醇-麦康凯培养基(SMAC)上,E.coli O157:H7与阪崎肠杆菌、普通变形杆菌、奇异变性杆菌以及铜绿假单胞菌都显无色而难以区分,而non-O157E.coli与产气肠杆菌、阴沟肠杆菌、肺炎克雷伯氏菌、鼠伤寒沙门氏菌、肠炎沙门氏菌以及伤寒沙门氏菌都显红色而难以区分,并且随着培养时间的延长,也会导致部分non-O157E.coli菌株红色褪去。由此可见,对于更多的E.coli O157、non-O157E.coli以及非大肠杆菌,现有的市售培养基TBX琼脂、CHROMagarE.coli、CHROMagar O157和山梨醇-麦康凯培养基(SMAC)均不能给出良好的检测特异性。
以上仅是本发明的优选实施方式,应当指出的是,上述优选实施方式不应视为对本发明的限制,本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说,在不脱离本发明的精神和范围内,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基,其特征在于,所述的显色培养基含有两种能产生无扩散性的不同颜色信号的显色底物和一种不显色但能产生无扩散性的非蓝色荧光信号的荧光底物,所述的显色底物和荧光底物中除了含有β-葡萄糖醛酸苷酶底物和β-半乳糖苷酶底物外,还至少含有β-葡萄糖苷酶底物、β-N-乙酰氨基葡萄糖苷酶底物、纤维二糖苷酶底物这三种所产颜色或荧光信号相同或相近底物中的一种底物。
2.根据权利要求1所述的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基,其特征在于,所述的显色底物为基于3-吲哚酚衍生物的吲哚酚基显色底物,所述的3-吲哚酚衍生物的结构如下式(I)所示:
3.根据权利要求2所述的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基,其特征在于,所述的基于3-吲哚酚衍生物的吲哚酚基显色底物为5-溴-4-氯-3-吲哚基-β-D-葡萄糖苷、5-溴-6-氯-3-吲哚基-β-D-半乳糖苷、5-溴-4-氯-3-吲哚基-β-D-葡萄糖醛酸苷环己胺盐或5-溴-6-氯-3-吲哚基-β-D-葡萄糖醛酸苷环己胺盐。
4.根据权利要求1所述的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基,其特征在于,所述的荧光底物为基于2-(苯并噻唑-2'-基)苯酚衍生物的荧光底物,所述的2-(苯并噻唑-2'-基)苯酚衍生物的结构如下式(2)所示:
5.根据权利要求4所述的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基,其特征在于,所述的基于2-(苯并噻唑-2'-基)苯酚衍生物的的荧光底物为2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖醛酸苷、2-(苯并噻唑-2'-基)-4-溴苯基-β-D-半乳糖苷或2-(苯并噻唑-2'-基)-4-溴苯基-β-D-葡萄糖苷。
6.根据权利要求1所述的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基,其特征在于,所述的显色培养基中显色底物和荧光底物的浓度均为0.01~0.30g/L。
7.根据权利要求6所述的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基,其特征在于,所述的显色培养基还含有十二烷基硫酸钠0.05~0.25g/L和/或胆盐0~1.50g/L、蛋白胨1~20g/L、酵母提取粉0~6g/L、氯化钠4~8g/L、琼脂10~20g/L、异丙基-β-D-硫代吡喃半乳糖苷0.01~0.10g/L。
8.根据权利要求7所述的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基,其特征在于,所述的显色培养基还含有乳糖、水杨苷或纤维二糖0.01~0.15g/L。
9.根据权利要求7或8所述的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基,其特征在于,所述的显色培养基还含有吐温80 0~0.60mL/L或者曲拉通X-100 0~0.20mL/L。
10.根据权利要求1所述的用于检测大肠杆菌O157与非O157大肠杆菌的显色培养基,其特征在于,所述的β-葡萄糖醛酸苷酶底物为羧酸形式或羧酸盐形式。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201811457345.4A CN110042142A (zh) | 2018-11-30 | 2018-11-30 | 一种用于检测大肠杆菌o157与非o157大肠杆菌的显色培养基 |
PCT/CN2019/115520 WO2020108237A1 (zh) | 2018-11-30 | 2019-11-05 | 一种用于检测大肠杆菌o157与非o157大肠杆菌的显色培养基 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201811457345.4A CN110042142A (zh) | 2018-11-30 | 2018-11-30 | 一种用于检测大肠杆菌o157与非o157大肠杆菌的显色培养基 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN110042142A true CN110042142A (zh) | 2019-07-23 |
Family
ID=67273262
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201811457345.4A Pending CN110042142A (zh) | 2018-11-30 | 2018-11-30 | 一种用于检测大肠杆菌o157与非o157大肠杆菌的显色培养基 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN110042142A (zh) |
WO (1) | WO2020108237A1 (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020108237A1 (zh) * | 2018-11-30 | 2020-06-04 | 广东省微生物研究所(广东省微生物分析检测中心) | 一种用于检测大肠杆菌o157与非o157大肠杆菌的显色培养基 |
WO2022095576A1 (zh) * | 2020-11-09 | 2022-05-12 | 山东博科生物产业有限公司 | 一种新型N-乙酰-β-D氨基葡萄糖苷酶检测试剂 |
Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5846761A (en) * | 1992-10-07 | 1998-12-08 | Rambach; Alain | Culture medium for the detection of E. coli and process for its use |
US6136554A (en) * | 1995-06-07 | 2000-10-24 | Biolog, Inc. | Microbiological media for isolation and indentification of enteric pathogens such as E. coli and salmonella |
US20070254326A1 (en) * | 2004-09-16 | 2007-11-01 | Biomerieux | Method for Detecting Streptococcus Agalactiae Using Esterase Activity |
CN101186894A (zh) * | 2007-07-31 | 2008-05-28 | 深圳市计量质量检测研究院 | 阪崎肠杆菌选择性分离培养基 |
CN101631873A (zh) * | 2007-02-08 | 2010-01-20 | 生物梅里埃公司 | 细菌检测和/或鉴定培养基 |
JP2011019462A (ja) * | 2009-07-17 | 2011-02-03 | Eiken Chemical Co Ltd | 腸管出血性大腸菌o157、o26、o111選択分離培地 |
CN102311990A (zh) * | 2011-09-16 | 2012-01-11 | 广州绿洲生化科技有限公司 | 一种大肠菌群的显色培养基及其快速检测卡 |
CN102424832A (zh) * | 2011-11-23 | 2012-04-25 | 广东环凯微生物科技有限公司 | 一种用于检测大肠杆菌o157:h7的显色培养基 |
CN104388521A (zh) * | 2014-11-24 | 2015-03-04 | 苏州嘉禧萝生物科技有限公司 | 用于检测大肠杆菌o157:h7的显色培养基 |
US20160046976A1 (en) * | 2013-04-03 | 2016-02-18 | bioMérieux | Use of at least one chromogenic and/or fluorogenic phosphatase substrate for the detection and/or enumeration of enterobacteria in a sample |
CN106432369A (zh) * | 2016-09-08 | 2017-02-22 | 广东省微生物研究所 | 一种基于吲哚酚衍生物、2‑(苯并噻唑‑2′‑基)苯酚衍生物的糖苷的合成方法 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110042142A (zh) * | 2018-11-30 | 2019-07-23 | 广东省微生物研究所(广东省微生物分析检测中心) | 一种用于检测大肠杆菌o157与非o157大肠杆菌的显色培养基 |
-
2018
- 2018-11-30 CN CN201811457345.4A patent/CN110042142A/zh active Pending
-
2019
- 2019-11-05 WO PCT/CN2019/115520 patent/WO2020108237A1/zh unknown
Patent Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5846761A (en) * | 1992-10-07 | 1998-12-08 | Rambach; Alain | Culture medium for the detection of E. coli and process for its use |
US6136554A (en) * | 1995-06-07 | 2000-10-24 | Biolog, Inc. | Microbiological media for isolation and indentification of enteric pathogens such as E. coli and salmonella |
US20070254326A1 (en) * | 2004-09-16 | 2007-11-01 | Biomerieux | Method for Detecting Streptococcus Agalactiae Using Esterase Activity |
CN101631873A (zh) * | 2007-02-08 | 2010-01-20 | 生物梅里埃公司 | 细菌检测和/或鉴定培养基 |
CN101186894A (zh) * | 2007-07-31 | 2008-05-28 | 深圳市计量质量检测研究院 | 阪崎肠杆菌选择性分离培养基 |
JP2011019462A (ja) * | 2009-07-17 | 2011-02-03 | Eiken Chemical Co Ltd | 腸管出血性大腸菌o157、o26、o111選択分離培地 |
CN102311990A (zh) * | 2011-09-16 | 2012-01-11 | 广州绿洲生化科技有限公司 | 一种大肠菌群的显色培养基及其快速检测卡 |
CN102424832A (zh) * | 2011-11-23 | 2012-04-25 | 广东环凯微生物科技有限公司 | 一种用于检测大肠杆菌o157:h7的显色培养基 |
US20160046976A1 (en) * | 2013-04-03 | 2016-02-18 | bioMérieux | Use of at least one chromogenic and/or fluorogenic phosphatase substrate for the detection and/or enumeration of enterobacteria in a sample |
CN104388521A (zh) * | 2014-11-24 | 2015-03-04 | 苏州嘉禧萝生物科技有限公司 | 用于检测大肠杆菌o157:h7的显色培养基 |
CN106432369A (zh) * | 2016-09-08 | 2017-02-22 | 广东省微生物研究所 | 一种基于吲哚酚衍生物、2‑(苯并噻唑‑2′‑基)苯酚衍生物的糖苷的合成方法 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020108237A1 (zh) * | 2018-11-30 | 2020-06-04 | 广东省微生物研究所(广东省微生物分析检测中心) | 一种用于检测大肠杆菌o157与非o157大肠杆菌的显色培养基 |
WO2022095576A1 (zh) * | 2020-11-09 | 2022-05-12 | 山东博科生物产业有限公司 | 一种新型N-乙酰-β-D氨基葡萄糖苷酶检测试剂 |
Also Published As
Publication number | Publication date |
---|---|
WO2020108237A1 (zh) | 2020-06-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR100517757B1 (ko) | 에스큘레틴유도체 | |
Manafi et al. | Comparative evaluation of different chromogenic/fluorogenic media for detecting Escherichia coli O157: H7 in food | |
EP1196624B1 (en) | Test media for identification and differentiation of enterobacteriaceae | |
US8883441B2 (en) | Method for detecting and counting micro-organisms in a sample | |
CN110042142A (zh) | 一种用于检测大肠杆菌o157与非o157大肠杆菌的显色培养基 | |
CN102603524B (zh) | 一种醌类衍生物及其制备方法与作为抗菌剂的应用 | |
CN102827918A (zh) | 一种用于检测沙门氏菌的显色培养基 | |
US7563592B2 (en) | Alizarin-based chromogenic substrates, their uses and composition containing same | |
EP1874951B1 (en) | Test media and method for identification and differentiation of bacteria | |
CN101186894B (zh) | 阪崎肠杆菌选择性分离培养基 | |
Tutenel et al. | Sensitivity of methods for the isolation of Escherichia coli O157 from naturally infected bovine faeces | |
James et al. | Note: Cyclohexenoesculetin‐β‐D‐glucoside: a new substrate for the detection of bacterial β‐D‐glucosidase | |
JP5751658B2 (ja) | 腸管出血性大腸菌o157、o26、o111選択分離培地 | |
Chan et al. | Comparative study of colorimetric DNA hybridization method and conventional culture procedure for detection of Salmonella in foods | |
CN109810157B (zh) | 一种β-葡萄糖醛酸苷酶沉淀型荧光底物合成方法 | |
EP1224196B1 (fr) | Substrat enzymatique, procede de synthese et utilisations | |
EP3066107B1 (en) | Chromogenic substrates for beta-d-glucuronidase activity and use thereof for microbial detection | |
CN106432369A (zh) | 一种基于吲哚酚衍生物、2‑(苯并噻唑‑2′‑基)苯酚衍生物的糖苷的合成方法 | |
JP4472078B2 (ja) | 菌の分離・検出方法 | |
CN113980072B (zh) | 一种β-葡萄糖苷酶荧光底物CFMU-Glu及其制备方法和应用 | |
KR100353143B1 (ko) | 대장균 o157 : h7 검출용 고체 평판 배지 | |
JP5354564B2 (ja) | 大腸菌o26およびo157の同時検出培地ならびにその検出法 | |
Heuvelink | Culture media for the isolation of diarrhoeagenic Escherichia coli from foods | |
JP5826005B2 (ja) | 腸管出血性大腸菌検出用培地 | |
Elizaquível et al. | 75 Escherichia |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190723 |
|
RJ01 | Rejection of invention patent application after publication |