CN104406954A - Surface enhanced Raman spectra substrate for detecting avian influenza virus, and application thereof - Google Patents
Surface enhanced Raman spectra substrate for detecting avian influenza virus, and application thereof Download PDFInfo
- Publication number
- CN104406954A CN104406954A CN201410697470.8A CN201410697470A CN104406954A CN 104406954 A CN104406954 A CN 104406954A CN 201410697470 A CN201410697470 A CN 201410697470A CN 104406954 A CN104406954 A CN 104406954A
- Authority
- CN
- China
- Prior art keywords
- avian influenza
- influenza virus
- enhanced raman
- surface enhanced
- formula
- 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.)
- Granted
Links
- 241000712461 unidentified influenza virus Species 0.000 title claims abstract description 71
- 238000001237 Raman spectrum Methods 0.000 title claims abstract description 13
- 239000000758 substrate Substances 0.000 title abstract description 27
- 239000002245 particle Substances 0.000 claims abstract description 37
- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 claims abstract description 34
- 239000010931 gold Substances 0.000 claims abstract description 25
- 238000001514 detection method Methods 0.000 claims abstract description 24
- 229910052737 gold Inorganic materials 0.000 claims abstract description 20
- 230000008878 coupling Effects 0.000 claims description 34
- 238000010168 coupling process Methods 0.000 claims description 34
- 238000005859 coupling reaction Methods 0.000 claims description 34
- 238000001069 Raman spectroscopy Methods 0.000 claims description 31
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 19
- 150000001875 compounds Chemical class 0.000 claims description 18
- 238000012360 testing method Methods 0.000 claims description 18
- 229910052739 hydrogen Inorganic materials 0.000 claims description 10
- 239000001257 hydrogen Substances 0.000 claims description 8
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 7
- 238000001556 precipitation Methods 0.000 claims description 6
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 4
- 125000000738 acetamido group Chemical group [H]C([H])([H])C(=O)N([H])[*] 0.000 claims description 4
- 150000002431 hydrogen Chemical class 0.000 claims description 4
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 2
- SHZGCJCMOBCMKK-PQMKYFCFSA-N L-Fucose Natural products C[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O SHZGCJCMOBCMKK-PQMKYFCFSA-N 0.000 claims description 2
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 claims description 2
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 claims description 2
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims description 2
- 230000003595 spectral effect Effects 0.000 abstract description 11
- 241000700605 Viruses Species 0.000 abstract description 5
- 238000002474 experimental method Methods 0.000 abstract description 4
- 238000000479 surface-enhanced Raman spectrum Methods 0.000 abstract description 3
- 239000012535 impurity Substances 0.000 abstract description 2
- 238000001228 spectrum Methods 0.000 abstract 1
- 230000003612 virological effect Effects 0.000 description 9
- 239000000203 mixture Substances 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 238000005516 engineering process Methods 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 241001473386 H9N2 subtype Species 0.000 description 5
- 206010022000 influenza Diseases 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 229910021642 ultra pure water Inorganic materials 0.000 description 5
- 239000012498 ultrapure water Substances 0.000 description 5
- 241000287828 Gallus gallus Species 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 241001500351 Influenzavirus A Species 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 206010064097 avian influenza Diseases 0.000 description 2
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 235000019800 disodium phosphate Nutrition 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 229910000510 noble metal Inorganic materials 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012372 quality testing Methods 0.000 description 2
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 2
- 229940038773 trisodium citrate Drugs 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 101710154606 Hemagglutinin Proteins 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
- 208000002979 Influenza in Birds Diseases 0.000 description 1
- 239000012901 Milli-Q water Substances 0.000 description 1
- 241001183012 Modified Vaccinia Ankara virus Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 1
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 1
- 101150105115 PA gene Proteins 0.000 description 1
- 101710176177 Protein A56 Proteins 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 238000009361 aviculture Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000007769 metal material Substances 0.000 description 1
- 238000010905 molecular spectroscopy Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000001686 rotational spectrum Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 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 1
- 239000002689 soil Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 229910052723 transition metal Inorganic materials 0.000 description 1
- 150000003624 transition metals Chemical class 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000001845 vibrational spectrum Methods 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 230000004304 visual acuity Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a surface enhanced Raman spectra substrate for detecting an avian influenza virus and application of the surface enhanced Raman spectra substrate. The application provided by the invention specifically relates to the application of nano-gold particles coupled with alpha (2 and 3) sialyloligosaccharide in the surface enhanced Raman spectra substrate for detecting the avian influenza virus. Experiments show that the substrate can specifically capture the avian influenza virus in a sample to be detected; the enrichment of avian influenza viruses in the sample can be realized through centrifugal collection, and then the enriched avian influenza viruses are used for SERS (Surface Enhanced Raman Scattering) spectra detection. An interference wave crest caused by sample impurities is effectively removed because of the enrichment of the viruses, and a spectral signal can also be effectively amplified by the nano-gold particles, so that the substrate can be used for the SERS spectra detection of the avian influenza virus. The substrate has a great significance to the rapid detection and identification of the avian influenza virus.
Description
Technical field
The invention belongs to field of virus detection, relate to a kind of the Raman spectrum base thing and the application thereof that detect avian influenza virus.
Background technology
Nineteen twenty-eight India physicist C .V.Raman finds after monochromatic light incides sample, molecule in sample can produce scattering to incident light, scattered light is except having the spectral line identical with incident light frequency, also there is the spectral line be subjected to displacement, we claim this scattering to be Raman (Raman) scattering.Raman spectrum belongs to molecule rotation and vibrational spectrum category, and existing molecular structure information obtains mostly from molecular spectroscopy.Therefore Raman spectroscopy has important using value in fields such as physics, chemistry, life science, medical science, material science.But spontaneous Raman scattering signal is very weak, because which limit its application.When some molecular adsorption in or near some coarse metal surface time, their Raman signal intensity can increase by 10
2-10
7doubly, the phenomenon that this Raman signal intensity significantly strengthens is called Surface enhanced raman spectroscopy (Surface-enhanced Raman Scattering, SERS) effect.The features such as SERS spectrum is little with its high resolving power, high sensitivity, solution interference, good stability obtain application in a lot of field.
Experiment proves, occurs that the metal material of SERS effect phenomenon only has a few, have been found that at present can produce SERS effect have noble metal gold, silver, copper etc. and some conventional transition metal (as iron, cobalt, nickel) etc.Metal electrode is the SERS substrate technology of preparing studied the earliest, is also current technology relative maturity, usable range substrate technology of preparing comparatively widely.Noble metal sol particle method is also a kind of comparatively conventional SERS substrate preparation method.This sample advantage to be placed in atmosphere the long period, and more stable, has more wide usable range than metal electrode active substrate.The selection of good substrate and preparation are the prerequisites obtaining SERS signal.
The RNA virus of avian influenza virus to be a kind of with wild fowl be natural host, can infect poultry, be affect China's aviculture virus the most serious, cause economic loss to reach tens billion of unit every year.Avian influenza virus also can infect people once in a while, the large influenza of Spain of 1918 causes the death more than 4,000 ten thousand people, this viral source of rear proof is in avian influenza virus, and 1997 and the H5N1 virus of 2003 and the H7N9 virus of 2013 cause China up to a hundred people dead, are also all avian influenza virus.Visible, avian influenza virus forms threat greatly to domestic fowl farming aquaculture and human health.Surface enhanced raman spectroscopy technology can be applied to the detection of avian influenza virus, but mostly being ight soil or cloacal swab due to clinical sample to be detected, sample composition is complicated, and viral level is lower, therefore Valid peak is covered in the assorted summit of SERS spectrum, causes false negative result.
Summary of the invention
An object of the present invention is to provide a kind of novelty teabag that coupling has the nanogold particle of α (2,3) sialyloligosaccharide.
Coupling provided by the present invention has α (2,3) novelty teabag of the nanogold particle of sialyloligosaccharide, be specially coupling and have the nanogold particle of α (2,3) sialyloligosaccharide as the application for detecting in the Raman spectrum base thing of avian influenza virus.
Another object of the present invention is to provide a kind of kit utilizing Surface enhanced raman spectroscopy to detect or whether contain avian influenza virus in auxiliary detection testing sample.
The kit utilizing Surface enhanced raman spectroscopy to detect or whether contain avian influenza virus in auxiliary detection testing sample provided by the present invention, has nanogold particle and the instructions of α (2,3) sialyloligosaccharide containing coupling;
Following content is recorded in described instructions:
There is the nanogold particle of α (2,3) sialyloligosaccharide to detect the method whether containing avian influenza virus in testing sample with described coupling, comprise the steps:
(a1) experimental group: described Raman spectrum base thing is mixed with the volume ratio of testing sample according to 10:1,20-25 DEG C (as 25 DEG C) leave standstill 10min; The centrifugal 30min of 8000g, get precipitation PBS (pH7.4) resuspended after carry out Surface enhanced raman spectroscopy detection;
Wherein, " centrifugal-PBS is resuspended " step can repetitive operation once; The amount of the PBS that resuspended precipitation is used all can be 10 times of volumetric usage of described testing sample.
The solvent of described PBS is water, solute and concentration as follows: 0.274M sodium chloride, 0.005M potassium chloride, 0.012M sodium hydrogen phosphate, 0.004M potassium dihydrogen phosphate, pH7.4.
Control group: compared with described experimental group, only replaces with the water of equivalent by described testing sample, all the other are all identical;
(a2) according to the testing result of step (a1), according to determining in described testing sample whether containing avian influenza virus as follows: if the surface enhanced Raman spectroscopic signals of described experimental group is better than the surface enhanced Raman spectroscopic signals of described control group, then in described testing sample containing or candidate contain avian influenza virus; If the surface enhanced Raman spectroscopic signals of described experimental group is not better than the surface enhanced Raman spectroscopic signals of described control group, then in described testing sample not containing or candidate not containing avian influenza virus
In actual applications, judge that the power of the surface enhanced Raman spectroscopic signals of described experimental group and the surface enhanced Raman spectroscopic signals of described control group specifically can be determined according to the peak height of Surface enhanced raman spectroscopy figure.Specific as follows: if the peak height of the Surface enhanced raman spectroscopy figure of described experimental group is higher than the peak height of the Surface enhanced raman spectroscopy figure of control group described under identical Raman shift, then the surface enhanced Raman spectroscopic signals of described experimental group is better than the surface enhanced Raman spectroscopic signals of described control group; Otherwise then the surface enhanced Raman spectroscopic signals of described experimental group is not better than the surface enhanced Raman spectroscopic signals of described control group.
In described (a1), the parameter of carrying out when described Surface enhanced raman spectroscopy detects is specific as follows: it is 80mW that Raman measures power, and wavelength is 785nm, and integral time is 20s;
In the present invention, the instrument carrying out in step (a1) adopting when described Surface enhanced raman spectroscopy detects is specially RamTracer-200-HS high sensitivity laser Raman spectrometer.
In application described above or kit, described coupling has the nanogold particle of α (2,3) sialyloligosaccharide for being connected to the gold nano grain of compound shown in compound and formula II shown in formula I by S-Au covalent bond on surface:
Formula I:
Formula II:HO (CH
2cH
2o)
m(CO)
n(CH
2)
k(CH)
psR;
In described formula I and described formula II, X is the integer of 1-3, and m is the integer of 0-6, and n is the integer of 0-1, and k is the integer of 0-11, p be 0 or 2, R be S or H, Ac be acetyl group, R
1for hydroxyl or acetamido, R
2for hydrogen or L-fucose, R
3for hydrogen or sulfuric ester, R
4for hydroxyl or acetamido.
In one embodiment of the invention, in described formula I and described formula II, X is 1, m be 3, n be 1, k be 4, p be 1, R is S, R
1for hydroxyl, R
2for hydrogen, R
3for hydrogen, R
4for hydroxyl.Specific as follows:
In described application or kit, the diameter of described nanogold particle can be 10-30nm.
In an embodiment of we's invention, the diameter of described nanogold particle is specially 10-15nm.
In the present invention, described nanogold particle specifically method in accordance with the following steps prepare:
(1) gold chloride is soluble in water, heating adds the trisodium citrate aqueous solution that concentration is 1g/100ml after boiling; The proportioning of described gold chloride, described water and described trisodium citrate aqueous solution is 50mg:500ml:8.7ml;
(2) be cooled to 20-25 DEG C after stirring continues to boil 35min, obtain described nanogold particle.
In described application or kit, forming described coupling has the mol ratio of compound and described gold nano grain shown in compound, described formula II shown in the described formula I of the nanogold particle of α (2,3) sialyloligosaccharide to can be 10000 ~ 80000:2000 ~ 16000:1.
In an embodiment of we's invention, forming described coupling has the mol ratio of compound and described gold nano grain shown in compound, described formula II shown in the described formula I of the nanogold particle of α (2,3) sialyloligosaccharide to be specially 40000:8000:1.
In the present invention, the method being connected compound shown in compound and described formula II shown in described formula I by S-Au covalent bond on described gold nano grain surface specifically can comprise the steps: by compound shown in compound shown in described formula I, described formula II and described gold nano grain according to mol ratio be 10000 ~ 80000:5000 ~ 40000:1 (concrete as 40000:20000:1) ratio mixing after in 20-25 DEG C (as 25 DEG C) stirring 24 hours.
The rotating speed of described stirring specifically can be 100 ~ 300 revs/min (as 200 revs/min).
In the process, the step of carrying out twice washing with water (as ultrapure water) is also comprised after described stirring.
In the present invention, above all described avian influenza virus all can be any one as follows: avian influenza virus A/Vietnam/1194/2004 (H5N1), avian influenza virus A/Chicken/Shanghai/441/2009 (H9N2), avian influenza virus A/Puerto Rico/8/34 (H1N1), avian influenza virus A/Anhui/1/2013 (H7N9).
The present invention, by α (2, the 3) sialyloligosaccharide of Prof. Du Yucang and nanogold particle covalent coupling, it can be used as the substrate of SERS spectral detection avian influenza virus.This substrate can be special the avian influenza virus of catching in measuring samples, by collected by centrifugation, the enrichment of avian influenza virus in sample can be realized, and then for SERS spectral detection.Because viral enrichment effectively eliminates the interference crest that sample impurity causes, and nanogold particle effectively can amplify spectral signal, and therefore this substrate can be used for the SERS spectral detection of avian influenza virus.This substrate is to the quick detection of avian influenza virus and differentiate significant.
Accompanying drawing explanation
Fig. 1 is that Surface enhanced raman spectroscopy detects avian influenza virus collection of illustrative plates.Wherein, 1 for have α (2 with coupling, 3) nanogold particle of sialyloligosaccharide detects avian influenza virus A/Vietnam/1194/2004 (H5N1) as substrate, 2 is with non-coupling α (2, 3) nanogold particle of the routine of sialyloligosaccharide detects avian influenza virus A/Vietnam/1194/2004 (H5N1) as substrate, 3 is that the coupling not adding viral sample has α (2, 3) nanogold particle of sialyloligosaccharide, 4 for have α (2 with coupling, 3) nanogold particle of sialyloligosaccharide detects human influenza virus A/California/04/2009 (H1N1) as substrate.
Fig. 2 adopts Surface enhanced raman spectroscopy to detect avian influenza virus Sample Flow Graph with substrate of the present invention.
Fig. 3 adopts Surface enhanced raman spectroscopy to detect different subtype avian influenza viruses collection of illustrative plates with substrate of the present invention.Wherein, 1 is avian influenza virus A/Chicken/Shanghai/441/2009 (H9N2), 2 is avian influenza virus A/Vietnam/1194/2004 (H5N1), 3 is avian influenza virus A/Puerto Rico/8/34 (H1N1), and 4 is avian influenza virus A/Anhui/1/2013 (H7N9).
Embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
RamTracer-200-HS high sensitivity laser Raman spectrometer (Ou Putusi optical nano Science and Technology Ltd.).Gold chloride, sodium citrate are purchased from traditional Chinese medicines group chemical reagent Beijing company limited.
Avian influenza virus A/Vietnam/1194/2004 (H5N1): be recorded in " Jarocka U; Sawicka R; Gora-Sochacka A; et al.An immunosensor based on antibody binding fragments attached togold nanoparticles for the detection of peptides derived from avian influenza hemagglutininH5.Sensors (Basel); 2014; 14 (9): 15714-15728. " literary composition, the public can obtain from Institute of Microorganism, Academia Sinica.
Avian influenza virus A/Chicken/Shanghai/441/2009 (H9N2): be recorded in " Liu Jianlong; Teng Qiaoyang; Fan Zhao; the Efficacy evaluation of et al.H9N2 subtype avian influenza virus cell inactivation vaccine. Chinese poultry resource; 2012; 34 (021): 24-27. " in a literary composition, the public can obtain from Institute of Microorganism, Academia Sinica.
Avian influenza virus A/Puerto Rico/8/34 (H1N1): be recorded in " Kamlangdee A; Kingstad-Bakke B; Anderson TK; et al.Broad Protection against Avian Influenza Virus by Using a ModifiedVaccinia Ankara Virus Expressing a Mosaic Hemagglutinin Gene.J Virol; 2014; 88 (22): 13300-13309. " literary composition, the public can obtain from Institute of Microorganism, Academia Sinica.
Avian influenza virus A/Anhui/1/2013 (H7N9): be recorded in " Si Y; Li J; Niu Y; et al.Entry Propertiesand Entry Inhibitors of a Human H7N9Influenza Virus.PLoS One; 2014; 9 (9): e107235. " literary composition, the public can obtain from Institute of Microorganism, Academia Sinica.
Human influenza virus A/California/04/2009 (H1N1): be recorded in " document: Sun Y; Xu Q; Shen Y; et al.Naturally occurring mutations in the PA gene are key contributors to increased virulence ofpandemic H1N1/09influenza virus in mice.J Virol; 2014; 88 (8): 4600-4604. " literary composition, the public can obtain from Institute of Microorganism, Academia Sinica.
The preparation of the Raman spectrum base thing of embodiment 1, detection avian influenza virus
One, the preparation of nanogold particle
50mg gold chloride is dissolved in 500ml ultrapure water, heating adds the citric acid three sodium solution (solvent is water) of 1% (1g/100ml) of 8.7ml after boiling, vigorous stirring is also cooled to room temperature (25 DEG C) after continuing to boil 35min, obtain the nanogold particle that particle diameter is about 10-15nm.
Two, the preparation of the nanogold particle of coupling α (2,3) sialyloligosaccharide
1, the preparation of α (2,3) sialic acid trisaccharide-short connection chain
Concrete preparation method is the embodiment 1 of the Chinese patent application of 201310495249.x (publication number is CN 103551562A) see application number.Obtain α (2,3) sialic acid trisaccharide-short connection chain as shown in following formula III and such as formula the short connection chain shown in IIII.
Formula III:
Formula IV:HO (CH
2cH
2o)
3cO (CH
2)
4cHSS
2, the preparation of the nanogold particle of coupling α (2,3) sialyloligosaccharide
Be that the embodiment 10 of the Chinese patent application of 201310495249.x (publication number is) operates see application number, specific as follows:
The gold nano grain that shown in formula III step 1 synthesized, shown in compound, formula IV prepared by compound and step one is in 25 DEG C of stirrings (200rpm) 24 hours after the ratio mixing of 40000:8000:1 according to mol ratio, is then 10mM (with nm of gold content meter) with being dissolved in ultrapure water to final concentration after milli-Q water twice.
Embodiment 2, Raman spectrum base quality testing survey the checking of avian influenza virus
The coupling obtained in embodiment 1 there is α (2,3) nanogold particle of sialyloligosaccharide is as substrate, mix from different influenza virus sample, carry out Surface enhanced raman spectroscopy detection, set up simultaneously and do not add the detection of viral sample only containing substrate and contrast, and select the nanogold particle of the non-coupling sialyloligosaccharide in embodiment 1 to carry out detection contrast.Specific embodiments is as follows:
1,4 1.5ml centrifuge tubes are got, 3 centrifuge tubes add the nano-Au solution that coupling that 100 μ l embodiments 1 obtain has α (2,3) sialyloligosaccharide respectively, are numbered 1,3,4, another one centrifuge tube adds the nm of gold of the non-coupling sialyloligosaccharide of 100 μ l, is numbered 2.Then 10 μ l avian influenza virus A/Vietnam/1194/2004 (H5N1) strain samples (10 are added at No. 1 pipe respectively
7virion/milliliter), No. 2 pipes add avian influenza virus A/Vietnam/1194/2004 (H5N1) the strain sample (10 preserved in 10 these laboratories of μ l
7virion/milliliter), No. 3 pipes add 10 μ l ultrapure waters in contrast, and No. 4 pipes add 10 μ l human influenza virus A/California/04/2009 (H1N1) strain samples (10
7virion/milliliter).
2, the potpourri room temperature (25 DEG C) being numbered 1,3,4 centrifuge tubes leaves standstill 10 minutes, then centrifugal 30 minutes of 10000rpm (being equivalent to 8000g), abandon supernatant, by precipitation 100 μ l PBS (formula: 0.274M sodium chloride, 0.005M potassium chloride, 0.012M sodium hydrogen phosphate, 0.004M potassium dihydrogen phosphate, pH7.4) resuspended.The potpourri room temperature (25 DEG C) being numbered 2 centrifuge tubes leaves standstill 10 minutes, and 4 samples are carried out Surface enhanced raman spectroscopy detection respectively, and Raman measures power 80mW, wavelength is 785nM, and integral time, 20s, measured the mean value of twice, obtain spectrogram, result as shown in Figure 1.
As seen from Figure 1, compared with the control group (managing for No. 3) substituting viral sample with equivalent ultrapure water, coupling of the present invention is adopted to have α (2,3) substrate that detects as surface-enhanced Raman of the nm of gold of sialyloligosaccharide, obviously can strengthen the spectral signal (No. 1 pipe) of avian influenza virus sample, the spectral signal (No. 4 pipes) of human influenza virus's sample can not be strengthened.And have α (2 with non-coupling, 3) nm of gold of sialyloligosaccharide is compared (No. 2 pipes), coupling has raman spectral signal during nm of gold group (No. 1 pipe) the detection avian influenza virus of α (2,3) sialyloligosaccharide stronger, and crest is more special.The Surface enhanced raman spectroscopy that demonstrating coupling of the present invention has the nm of gold of α (2,3) sialyloligosaccharide to may be used for avian influenza virus detects.
Embodiment 3, Raman spectrum base quality testing survey the application of different subtype avian influenza viruses
By in embodiment 1 obtain coupling have the nm of gold of α (2,3) sialyloligosaccharide as substrate respectively from different Influenza virus strain sample mix, carry out Surface enhanced raman spectroscopy detection, operation steps process flow diagram is as shown in Figure 2.Specific embodiments is as follows:
1,4 1.5ml centrifuge tubes are got, add 100 μ l couplings respectively and have α (2,3) nano-Au solution of sialyloligosaccharide, be numbered 1,2,3,4, then add 10 μ l avian influenza virus A/Chicken/Shanghai/441/2009 (H9N2) strain samples (10 at No. 1 pipe respectively
7virion/milliliter), No. 2 pipes add 10 μ l avian influenza virus A/Vietnam/1194/2004 (H5N1) strain samples (10
7virion/milliliter), No. 3 pipes add 10 μ l avian influenza virus A/Puerto Rico/8/34 (H1N1) strain samples (10
7virion/milliliter), No. 4 pipes add 10 μ l avian influenza virus A/Anhui/1/2013 (H7N9) strain samples (10
7virion/milliliter).
2, after potpourri mixing, room temperature (25 DEG C) leaves standstill 10 minutes, then centrifugal 30 minutes of 10000rpm (being equivalent to 8000g), abandon supernatant, by precipitation 100 μ l PBS (pH7.4, concrete formula sees above) resuspended, then centrifugal 30 minutes of 10000rpm (being equivalent to 8000g), abandons supernatant, again that precipitation is resuspended with 100 μ l PBS (pH7.4, concrete formula sees above).
3,4 samples are carried out Surface enhanced raman spectroscopy detection respectively, Raman measures power 80mw, and wavelength is 785nM, integral time 20s), measure the mean value of twice, obtain spectrogram.
Experiment arranges the control group substituting viral sample with equivalent ultrapure water simultaneously.
Result shows, and the surface enhanced Raman spectroscopic signals of 4 viral sample groups is all better than control group.The spectral signal of 4 viral sample is removed the background signal of pure water group in contrast, obtain result as shown in Figure 3.As seen from Figure 3, the substrate adopting coupling of the present invention to have the nm of gold of α (2,3) sialyloligosaccharide to detect as surface-enhanced Raman, to the avian influenza virus of different subtype, all there is enhancement effect, show the reliability of the present invention as substrate.The Surface enhanced raman spectroscopy that demonstrating coupling of the present invention has the nm of gold of α (2,3) sialyloligosaccharide to may be used for avian influenza virus detects.
Claims (9)
1. coupling has the nanogold particle of α (2,3) sialyloligosaccharide as the application for detecting in the Raman spectrum base thing of avian influenza virus.
2. utilize the kit whether containing avian influenza virus in Surface enhanced raman spectroscopy detection or auxiliary detection testing sample, have nanogold particle and the instructions of α (2,3) sialyloligosaccharide containing coupling;
Following content is recorded in described instructions:
There is the nanogold particle of α (2,3) sialyloligosaccharide to detect the method whether containing avian influenza virus in testing sample with described coupling, comprise the steps:
(a1) experimental group: described coupling had the nanogold particle of α (2,3) sialyloligosaccharide to mix with the volume ratio of described testing sample according to 10:1,20-25 DEG C of standing 10min; The centrifugal 30min of 8000g, get precipitation PBS resuspended after carry out Surface enhanced raman spectroscopy detection;
Control group: compared with described experimental group, only replaces with the water of equivalent by described testing sample, all the other are all identical;
(a2) according to the testing result of step (a1), according to determining in described testing sample whether containing avian influenza virus as follows: if the surface enhanced Raman spectroscopic signals of described experimental group is better than the surface enhanced Raman spectroscopic signals of described control group, then in described testing sample containing or candidate contain avian influenza virus; If the surface enhanced Raman spectroscopic signals of described experimental group is not better than the surface enhanced Raman spectroscopic signals of described control group, then in described testing sample not containing or candidate not containing avian influenza virus.
3. kit according to claim 2, is characterized in that: in described (a1), and the parameter of carrying out when described Surface enhanced raman spectroscopy detects is as follows: it is 80mW that Raman measures power, and wavelength is 785nm, and integral time is 20s;
4. application according to claim 1 or the kit described in Claims 2 or 3, it is characterized in that: described coupling has the nanogold particle of α (2,3) sialyloligosaccharide for being connected to the gold nano grain of compound shown in compound and formula II shown in formula I by S-Au covalent bond on surface:
Formula I:
Formula II:HO (CH
2cH
2o)
m(CO)
n(CH
2)
k(CH)
psR;
In described formula I and described formula II, X is the integer of 1-3, and m is the integer of 0-6, and n is the integer of 0-1, and k is the integer of 0-11, p be 0 or 2, R be S or H, Ac be acetyl group, R
1for hydroxyl or acetamido, R
2for hydrogen or L-fucose, R
3for hydrogen or sulfuric ester, R
4for hydroxyl or acetamido.
5. application according to claim 4 or kit, is characterized in that: in described formula I and described formula II, and X is 1, m be 3, n be 1, k be 4, p be 1, R is S, R
1for hydroxyl, R
2for hydrogen, R
3for hydrogen, R
4for hydroxyl.
6., according to described application arbitrary in claim 1-5 or kit, it is characterized in that: the diameter of described nanogold particle is 10-30nm.
7. application according to claim 6 or kit, is characterized in that: the diameter of described nanogold particle is 10-15nm.
8. according to described application arbitrary in claim 1-7 or kit, it is characterized in that: the mol ratio forming compound and described gold nano grain shown in compound, described formula II shown in described formula I that described coupling has a nanogold particle of α (2,3) sialyloligosaccharide is 10000 ~ 80000:2000 ~ 16000:1.
9. application according to claim 8 or kit, it is characterized in that: the mol ratio forming compound and described gold nano grain shown in compound, described formula II shown in described formula I that described coupling has a nanogold particle of α (2,3) sialyloligosaccharide is 40000:8000:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410697470.8A CN104406954B (en) | 2014-11-26 | 2014-11-26 | A kind of Raman spectrum base thing for detecting avian influenza virus and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410697470.8A CN104406954B (en) | 2014-11-26 | 2014-11-26 | A kind of Raman spectrum base thing for detecting avian influenza virus and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104406954A true CN104406954A (en) | 2015-03-11 |
| CN104406954B CN104406954B (en) | 2017-06-30 |
Family
ID=52644601
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410697470.8A Expired - Fee Related CN104406954B (en) | 2014-11-26 | 2014-11-26 | A kind of Raman spectrum base thing for detecting avian influenza virus and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104406954B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105603125A (en) * | 2016-03-09 | 2016-05-25 | 华南农业大学 | Detection reagent kit for rapidly detecting poultry disease viruses and detection method of detection reagent kit |
| CN106589014A (en) * | 2016-12-05 | 2017-04-26 | 中国科学院微生物研究所 | Sialyloligosaccharide-magnetic nanoparticles as well as preparation method and application thereof |
| CN109060766A (en) * | 2018-09-27 | 2018-12-21 | 珠海彩晶光谱科技有限公司 | A kind of measurement method and detection device of the concentration in body fluid based on Surface enhanced Raman spectroscopy |
| CN109811089A (en) * | 2019-01-02 | 2019-05-28 | 南京邮电大学 | A kind of influenza virus gene segment SERS detection kit and preparation method thereof |
| CN112315056A (en) * | 2020-10-16 | 2021-02-05 | 杭州医学院 | Special detection mask and method of Raman immune probe for respiratory tract virus collection and detection |
| CN113252640A (en) * | 2021-06-03 | 2021-08-13 | 季华实验室 | Rapid virus screening and detecting method |
| CN113984747A (en) * | 2021-10-21 | 2022-01-28 | 南京理工大学 | Method for modifying sialic acid on surface of gold nano array |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1850972A (en) * | 2006-05-17 | 2006-10-25 | 吴培星 | Sialic acid oligose-chitosan complex, and its preparing method and use |
| US20080297789A1 (en) * | 2002-01-10 | 2008-12-04 | Chemimage Corporation | Raman Spectral Analysis of Pathogens |
| KR20090030767A (en) * | 2007-09-21 | 2009-03-25 | 재단법인서울대학교산학협력재단 | Detection method of pathogenic agents and single nucleotide polymorphism using dna hybridization by surface-enhanced raman scattering using unmodified gold nanoparticles |
| CN103551562A (en) * | 2013-10-21 | 2014-02-05 | 中国科学院微生物研究所 | Sialyloligosaccharide-gold nano particle and preparation method and applications thereof |
-
2014
- 2014-11-26 CN CN201410697470.8A patent/CN104406954B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080297789A1 (en) * | 2002-01-10 | 2008-12-04 | Chemimage Corporation | Raman Spectral Analysis of Pathogens |
| CN1850972A (en) * | 2006-05-17 | 2006-10-25 | 吴培星 | Sialic acid oligose-chitosan complex, and its preparing method and use |
| KR20090030767A (en) * | 2007-09-21 | 2009-03-25 | 재단법인서울대학교산학협력재단 | Detection method of pathogenic agents and single nucleotide polymorphism using dna hybridization by surface-enhanced raman scattering using unmodified gold nanoparticles |
| CN103551562A (en) * | 2013-10-21 | 2014-02-05 | 中国科学院微生物研究所 | Sialyloligosaccharide-gold nano particle and preparation method and applications thereof |
Non-Patent Citations (4)
| Title |
|---|
| CHI-CHANG LIN ET AL.: "A filter-like AuNPs@MS SERS substrate for Staphylococcus aureus detection", 《BIOSENSORS AND BIOELECTRONICS》 * |
| ERDENE-OCHIR GANBOLD ET AL.: "Aggregation effects of gold nanoparticles for single-base mismatch detection in influenza A (H1N1) DNA sequences using fluorescence and Raman measurements", 《COLLOIDS AND SURFACES B：BIOINTERFACES》 * |
| 杨亚伟 等: "唾液酸寡糖-壳聚糖复合物的制备及其对禽流感病毒的吸附作用", 《中国兽医科学》 * |
| 钟耀刚 等: "流感病毒识别糖链受体分子机制的研究进展", 《生物化学与生物物理进展》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105603125A (en) * | 2016-03-09 | 2016-05-25 | 华南农业大学 | Detection reagent kit for rapidly detecting poultry disease viruses and detection method of detection reagent kit |
| CN105603125B (en) * | 2016-03-09 | 2019-07-12 | 华南农业大学 | A kind of detection kit and its detection method of quick detection poultry diease virus |
| CN106589014A (en) * | 2016-12-05 | 2017-04-26 | 中国科学院微生物研究所 | Sialyloligosaccharide-magnetic nanoparticles as well as preparation method and application thereof |
| CN109060766A (en) * | 2018-09-27 | 2018-12-21 | 珠海彩晶光谱科技有限公司 | A kind of measurement method and detection device of the concentration in body fluid based on Surface enhanced Raman spectroscopy |
| CN109811089A (en) * | 2019-01-02 | 2019-05-28 | 南京邮电大学 | A kind of influenza virus gene segment SERS detection kit and preparation method thereof |
| CN112315056A (en) * | 2020-10-16 | 2021-02-05 | 杭州医学院 | Special detection mask and method of Raman immune probe for respiratory tract virus collection and detection |
| CN113252640A (en) * | 2021-06-03 | 2021-08-13 | 季华实验室 | Rapid virus screening and detecting method |
| CN113984747A (en) * | 2021-10-21 | 2022-01-28 | 南京理工大学 | Method for modifying sialic acid on surface of gold nano array |
| CN113984747B (en) * | 2021-10-21 | 2024-04-05 | 南京理工大学 | Method for modifying sialic acid on surface of gold nano array |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104406954B (en) | 2017-06-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104406954A (en) | Surface enhanced Raman spectra substrate for detecting avian influenza virus, and application thereof | |
| CN103852460B (en) | Based on the method that how residual the magnetic nano fluorescent sensor detection of antibiotics of aptamers is | |
| CN103076316A (en) | Method for detecting trace amount of oxytetracycline by utilizing CdTe quantum dot fluorescent probe | |
| CN104316460B (en) | A kind of TiO 2the preparation method of-CdSe nano composite material photoelectricity biology sensor and application thereof | |
| CN105175417B (en) | There is one-dimensional organic semiconductor nanotube of fluorescence response and its preparation method and application to organic amine gas | |
| CN109125416A (en) | A kind of quinoa wheat bran total saposins extract and purification process | |
| CN1866012A (en) | Quantitative and quick immune detection method and special apparatus therefor | |
| CN103852436B (en) | A kind of high specific DNA hydrogel detection method to lead ion | |
| CN102879381B (en) | Method for detecting antibacterial drugs of furazolidone and furacilin through surface-enhanced raman spectroscopy | |
| CN103865519B (en) | A kind of preparation method of up-conversion luminescence nano-probe and the application in mensuration material anti-oxidant activity thereof | |
| Kim et al. | Mouse adaptation of influenza B virus increases replication in the upper respiratory tract and results in droplet transmissibility in ferrets | |
| CN102579861A (en) | Method for detecting quality of An'erning granules | |
| Ma et al. | N-doped carbon dots as a fluorescent probe for the sensitive and facile detection of carbamazepine based on the inner filter effect | |
| CN107515206A (en) | A kind of method of sulfur doping carbon quantum dot fluorescence sensitivity detection Norfloxacin | |
| CN102608090A (en) | Homogeneous phase virus immune-detecting method based on quantum dot | |
| CN104830996A (en) | Pretreatment method for detecting influenza virus based on antibody magnetic bead capture | |
| CN105331739A (en) | Prawn covert mortality nodavirus fluorescent quantitative RT-PCR detection method | |
| KR20170063000A (en) | Method of Detecting Virus Using Virus Specific Nucleic Acid Aptamer-Nanoparticle Complex | |
| CN206751855U (en) | A kind of kit for highly pathogenic H7 avian flu virus detections | |
| CN109187452A (en) | One kind nano material of urea formaldehyde containing chlorophenol and its preparation and application | |
| CN102279172B (en) | Nano probe chip for detecting hand-foot-and-mouth disease pathogen and application method thereof | |
| CN104597254A (en) | C-reactive protein quick-detecting method and C-reactive protein quick-detecting kit | |
| CN105525038A (en) | Newcastle disease virus strong/weak virulent one-step real-time fluorescence RT-PCR detection kit | |
| CN103667524B (en) | Fluorescent quantitative RT-PCR kit used for detection of Newcastle disease virus class I and application of fluorescent quantitative RT-PCR kit | |
| CN103173574B (en) | The method of SVCV is detected based on liquid-phase chip |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Liu Wenjun Inventor after: Li Xuebing Inventor after: Yang Limin Inventor after: Li Jing Inventor after: Jia Xiaojuan Inventor after: Sun Lei Inventor after: Liu Zhigang Inventor before: Liu Wenjun Inventor before: Li Xuebing Inventor before: Yang Limin Inventor before: Li Jing Inventor before: Jia Xiaojuan Inventor before: Sun Lei |
|
| CB03 | Change of inventor or designer information | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20170523 Address after: 100101 Beichen West Road, Beijing, No. 1 hospital, No. 3, No. Applicant after: INSTITUTE OF MICROBIOLOGY, CHINESE ACADEMY OF SCIENCES Applicant after: Kexing Gene Technology (Tianjin) Co.,Ltd. Address before: 100101 Beichen West Road, Beijing, No. 1 hospital, No. 3, No. Applicant before: Institute of Microbiology, Chinese Academy of Sciences Applicant before: Liu Zhigang Effective date of registration: 20170523 Address after: 100101 Beichen West Road, Beijing, No. 1 hospital, No. 3, No. Applicant after: INSTITUTE OF MICROBIOLOGY, CHINESE ACADEMY OF SCIENCES Applicant after: Liu Zhigang Address before: 100101 Beichen West Road, Beijing, No. 1 hospital, No. 3, No. Applicant before: Institute of Microbiology, Chinese Academy of Sciences |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20170630 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |