CN110218668A - 一种惰性载体沙门氏菌及其潜在应用 - Google Patents
一种惰性载体沙门氏菌及其潜在应用 Download PDFInfo
- Publication number
- CN110218668A CN110218668A CN201910424369.8A CN201910424369A CN110218668A CN 110218668 A CN110218668 A CN 110218668A CN 201910424369 A CN201910424369 A CN 201910424369A CN 110218668 A CN110218668 A CN 110218668A
- Authority
- CN
- China
- Prior art keywords
- salmonella
- inert carrier
- chicken
- serum
- bacterium
- 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
- 241000607142 Salmonella Species 0.000 title claims abstract description 57
- 210000002966 serum Anatomy 0.000 claims abstract description 48
- 241000287828 Gallus gallus Species 0.000 claims abstract description 47
- 241000894006 Bacteria Species 0.000 claims abstract description 42
- 230000004520 agglutination Effects 0.000 claims abstract description 39
- 238000001514 detection method Methods 0.000 claims abstract description 29
- 238000012360 testing method Methods 0.000 claims abstract description 29
- 239000000427 antigen Substances 0.000 claims abstract description 24
- 108091007433 antigens Proteins 0.000 claims abstract description 24
- 102000036639 antigens Human genes 0.000 claims abstract description 24
- 238000006243 chemical reaction Methods 0.000 claims abstract description 19
- 230000001580 bacterial effect Effects 0.000 claims abstract description 12
- 230000004523 agglutinating effect Effects 0.000 claims abstract description 10
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 244000144992 flock Species 0.000 claims abstract description 4
- 230000002068 genetic effect Effects 0.000 claims abstract description 3
- 241000271566 Aves Species 0.000 claims description 13
- 208000037386 Typhoid Diseases 0.000 claims description 12
- 201000008297 typhoid fever Diseases 0.000 claims description 12
- 208000015181 infectious disease Diseases 0.000 claims description 8
- 229920001817 Agar Polymers 0.000 claims description 7
- 239000008272 agar Substances 0.000 claims description 7
- 244000052769 pathogen Species 0.000 claims description 4
- 230000001717 pathogenic effect Effects 0.000 claims description 4
- 208000030852 Parasitic disease Diseases 0.000 claims description 3
- 208000036142 Viral infection Diseases 0.000 claims description 3
- 230000009385 viral infection Effects 0.000 claims description 3
- 238000012408 PCR amplification Methods 0.000 claims description 2
- 238000005345 coagulation Methods 0.000 claims description 2
- 230000015271 coagulation Effects 0.000 claims description 2
- 230000008685 targeting Effects 0.000 claims description 2
- 239000002609 medium Substances 0.000 claims 3
- 239000006142 Luria-Bertani Agar Substances 0.000 claims 2
- 239000003153 chemical reaction reagent Substances 0.000 claims 2
- 230000002906 microbiologic effect Effects 0.000 claims 1
- 241000607149 Salmonella sp. Species 0.000 abstract description 3
- 238000009629 microbiological culture Methods 0.000 abstract description 3
- 241000196324 Embryophyta Species 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 239000011521 glass Substances 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 238000003745 diagnosis Methods 0.000 description 5
- 206010012735 Diarrhoea Diseases 0.000 description 4
- 101150101078 fimW gene Proteins 0.000 description 4
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- 206010039438 Salmonella Infections Diseases 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 238000010876 biochemical test Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 206010039447 salmonellosis Diseases 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- GBWARTHIRIVTNI-PJHQGUKWSA-N (2s)-2,6-diaminohexanoic acid;(2r,3s,4r)-2,3,4,5-tetrahydroxypentanal Chemical compound NCCCC[C@H](N)C(O)=O.OC[C@@H](O)[C@H](O)[C@@H](O)C=O GBWARTHIRIVTNI-PJHQGUKWSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- KUWPCJHYPSUOFW-YBXAARCKSA-N 2-nitrophenyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1[N+]([O-])=O KUWPCJHYPSUOFW-YBXAARCKSA-N 0.000 description 1
- 241001485474 Adonis amurensis Species 0.000 description 1
- 101710186708 Agglutinin Proteins 0.000 description 1
- 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 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000589562 Brucella Species 0.000 description 1
- 208000027312 Bursal disease Diseases 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000208368 Euonymus alatus Species 0.000 description 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 description 1
- 101710146024 Horcolin Proteins 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- 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 description 1
- 101710189395 Lectin Proteins 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- -1 MR test Chemical compound 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products 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-PICCSMPSSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 101710179758 Mannose-specific lectin Proteins 0.000 description 1
- 101710150763 Mannose-specific lectin 1 Proteins 0.000 description 1
- 101710150745 Mannose-specific lectin 2 Proteins 0.000 description 1
- 208000006758 Marek Disease Diseases 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- 208000010359 Newcastle Disease Diseases 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 206010029719 Nonspecific reaction Diseases 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 241000606860 Pasteurella Species 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000000910 agglutinin Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 206010064097 avian influenza Diseases 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 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 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 229940099352 cholate Drugs 0.000 description 1
- 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 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000005354 coacervation Methods 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000037029 cross reaction Effects 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 201000002491 encephalomyelitis Diseases 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- NNFCIKHAZHQZJG-UHFFFAOYSA-N potassium cyanide Chemical compound [K+].N#[C-] NNFCIKHAZHQZJG-UHFFFAOYSA-N 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229940082569 selenite Drugs 0.000 description 1
- MCAHWIHFGHIESP-UHFFFAOYSA-L selenite(2-) Chemical compound [O-][Se]([O-])=O MCAHWIHFGHIESP-UHFFFAOYSA-L 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 208000004441 taeniasis Diseases 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/42—Salmonella
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/255—Salmonella (G)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一种惰性载体沙门氏菌及其潜在应用,其有望开发成一种新型惰性载体菌,可应用于简便快速检测抗原或感染抗体的间接凝集试验检测方法的开发。所述惰性载体沙门氏菌目前已保藏于中国普通微生物菌种保藏管理中心(CGMCC),保藏地址为中国北京,保藏编号为CGMCCNo.17340,保藏日期为2019年3月18日,分类命名为沙门氏菌(Salmonella sp.),菌株代号S9。该沙门氏菌分离自健康鸡群,其惰性载体特性是在较高的工作浓度下的细菌数量与不同遗传背景的多种鸡源血清不发生任何肉眼可见的凝集反应,即与鸡源血清不产生非特异性凝集反应,我们称该沙门氏菌为惰性载体沙门氏菌。本发明为开发新型简便快速间接凝集检测方法提供了一种惰性载体菌和其潜在应用。
Description
发明领域
本发明隶属生物医学技术检测领域,涉及一种具有惰性载体特性的沙门氏菌及其分离和鉴定过程和潜在应用,揭示了这种惰性载体沙门氏菌在工作浓度细菌数量下与鸡源血清不发生非特异性凝集反应的特性,该分离株可作为一种惰性载体菌在简便快速检测抗原或感染抗体的间接凝集试验检测方法的开发中具有潜在应用前景。
背景技术
凝集试验是目前医学、兽医临床使用较为广泛的一种经典的疫病免疫学快速诊断方法,其原理是细菌、红细胞等颗粒性抗原结合相应的抗体后,当有电解质存在时,抗原颗粒出现相互凝集凝聚现象,形成凝集小块或颗粒,可通过肉眼观察。我们将参与反应的抗原称为凝集原,抗体称为凝集素。平板凝集试验是凝集反应中是目前应用较为广泛的一种定性方法,将诊断血清(含有已知抗体)与待检菌悬液各滴一滴于洁净的透明玻璃平板上,等体积轻轻混合后,室温等待2分钟内,如出现肉眼可见的颗粒状凝集,即为阳性反应,常用于细菌的鉴定和血清型的分型等。相反,也可用已知的诊断抗原来检测待检血清或全血中是否存在相应抗体,兽医临床上常用于布氏杆菌的玻板凝集反应和鸡白痢/禽伤寒沙门氏菌全血平板凝集试验等。值得注意的是,随着凝集试验的改良与发展,有一些研究将特异性抗原分子包被于乳胶颗粒等惰性载体检测血清抗体,这些在直接凝集反应的基础上发展起来的间接凝集反应技术,扩大了检测应用范围和增加了反应的敏感性。
凝集试验可适用于病原感染的快速诊断,具有众多优点,如:简便快捷、不需要额外昂贵仪器、成本低廉且可现场测试等,但是在实际应用中也逐渐暴露出一些弊端和技术瓶颈,如以检测鸡白痢/禽伤寒沙门氏菌感染的全血平板凝集抗原为例,该凝集抗原检测在实践应用中有一定的局限性,被报道存在多种交叉非特异性反应明显,各批次检测结果不稳定,弱阳性漏检等多种影响检测结果的因素,且该法仅对成年鸡群检测效果相对敏感,对雏鸡则可能存在较大检测误差。前期研究中,专利申请发明人所在实验室使用目前临床应用最广泛的商品化鸡白痢/禽伤寒沙门氏菌染色凝集抗原,分两次在不同时间检测同一批来自某鸡场200份血清时发现,两批的检测结果总符合率仅为81%,提示每批次检测结果并不稳定和一致。当与荷兰Biochek公司的沙门氏菌D群ELISA试剂盒对比检测结果时发现,检测结果总符合率仅为79.5%,阳性符合率(检出率或敏感性)为75.2-79.4%,阴性符合率为79.5-85.5%。上述检测结果和比较分析表明该商品化凝集抗原检测鸡白痢/禽伤寒沙门氏菌血清抗体时,敏感性、特异性和结果准确性均未达到较为理想的水平,提示目前商品化凝集抗原的检测结果存在一定程度或较为明显的假阳性错检和假阴性漏检。
凝集抗原检测结果准确度有待提高的根本原因在于目前应用于凝集试验的凝集抗原都为全菌抗原,其成分复合多种成分的细菌颗粒抗原,而非单一抗原,理论上说,这种多成分的抗原很大可能会与同科属、其他种属科细菌和其它成分的非特异性交叉反应,鉴于凝集抗原工作浓度和细菌数量,该弊端必然会影响甚至较为显著干扰检测和诊断结果,严重影响疫病净化效果和疫病净化工作的推进。因此,如果选择使用表达单一抗原因子的该惰性载体菌替代多成分的全菌抗原作为凝集抗原,在保留凝集反应结果直观,操作简便等优势的前提下,能精确和完善地提高凝集抗原反应的特异性,可有效改良病原感染的现场快速诊断方法,具有巨大的应用前景。
发明内容
本发明目的在于:针对目前医学、兽医临床上检测病原感染的经典型快速诊断技术—凝集试验的特异性、敏感性和检测结果准确性迫切需要提高和完善的情况,本发明分离鉴定了一株具有惰性载体特性的沙门氏菌,该惰性载体沙门氏菌与鸡源血清不发生非特异性凝集反应,有望在开发简便快速间接凝集检测方法中提供一种惰性载体菌和其潜在应用。
为实现上述目的,本发明提供如下技术方案:
一种惰性载体沙门氏菌S9,保藏于中国普通微生物菌种保藏管理中心(CGMCC),保藏地址为中国北京,保藏编号为CGMCC No.17340,保藏日期为2019年3月18日,分类命名为沙门氏菌(Salmonella sp.),菌株代号S9。
所述的载体菌(即惰性载体沙门氏菌)能在LB或XLD琼脂培养基中培养,培养方法如下:从保存的菌种中挑取少量划线于LB或XLD琼脂培养基中,培养温度为37℃,其中在LB琼脂平板中,37℃培养后可形成灰白色圆形菌落;在XLD琼脂平板中,37℃培养后可形成粉色圆形菌落。
本发明按照国家标准方法(GB 4789.4-2016)从临床上(健康鸡群)分离得到一株鸡源沙门氏菌,使用靶向沙门氏菌种属特异性fimW基因的引物鉴定沙门氏菌载体菌S9,PCR扩增结果与标准禽伤寒沙门菌U20株一致。利用生化试验对所述的载体菌进行生化鉴别,结果与标准禽伤寒沙门菌U20株的生化特征一致。
通过玻板凝集试验验证所述的载体菌(即惰性载体沙门氏菌)菌悬液不存在自凝现象,与不同遗传背景的多种鸡源血清不发生非特异性凝集反应,鸡源血清包括但不限于SPF鸡血清、健康鸡血清、鸡细菌感染阳性血清(如:禽大肠杆菌阳性血清、沙门氏菌阳性血清、葡萄球菌阳性血清和巴氏杆菌阳性血清等)、鸡寄生虫感染阳性血清(如鸡球虫病阳性血清、绦虫病阳性血清等)、鸡病毒性感染阳性血清(如:鸡新城疫阳性血清、禽流感阳性血清、鸡马立克氏病阳性血清、鸡传染性法氏囊病阳性血清、鸡减蛋下降综合征阳性血清、禽脑脊髓炎病毒感染阳性血清等)和各种品种鸡免疫血清,其检测的凝集反应结果都为阴性。
所述的惰性载体沙门氏菌在工作浓度下的细菌数量不与鸡白痢沙门氏菌感染SPF鸡血清,肠炎沙门氏菌感染SPF鸡血清发生非特异性凝集反应,而标准禽伤寒沙门菌U20株,鸡白痢沙门氏菌CVCC535(中国兽医微生物菌种保藏管理中心),肠炎沙门氏菌50336等均可与上述沙门氏菌感染SPF鸡血清发生凝集反应。
与现有技术相比较,本发明的特征优点在于:
本发明提供的一株沙门氏菌分离株S9,具有惰性载体特性,其在工作浓度下的细菌数量与鸡源血清不发生凝集反应。基于该惰性载体菌,可应用于简便快速检测抗原或感染抗体的间接凝集试验检测方法的开发,可创新性改进和完善现有凝集抗原抗体检测的凝集试验的特异性瓶颈,具有巨大的应用价值和市场前景。
附图说明
图1为沙门氏菌特异性引物fimW鉴定结果;
一种惰性载体沙门氏菌,其菌株代号为S9,保藏在中国普通微生物菌种保藏管理中心(CGMCC),保藏地址为中国北京,保藏编号为CGMCC No.17340,保藏日期为2019年3月18日,分类命名为沙门氏菌Salmonella sp.。
具体实施方式
下面结合实施例对本发明作进一步说明。实施例旨在对本发明进行举例描述,而非以任何形式对本发明进行限制。
实施例1:沙门氏菌S9的分离鉴定
360日龄健康蛋鸡于2016年10月18日采集自江苏无锡马山养禽集团蛋鸡场一养禽场2和4栋健康鸡群,将鸡放于超净台内,用75%酒精对提前去毛的鸡体表面进行消毒,无菌采集鸡的肝脏、脾脏、肠道等组织,放置于无菌玻璃培养皿充分研磨,将匀浆液吸入无菌试管中,并加入BPW中37℃过夜振摇培养。吸取培养液1mL接种于亚硒酸盐胱氨酸增菌液(SC)中进行选择性培养。用接种环吸取增菌液,划线接种于木糖赖氨酸脱氧胆盐(XLD)平板上,放置于37℃培养箱过夜培养,挑选可疑菌落。利用已报道的沙门氏菌种属引物fimW对分离株进行鉴定。取1mL上述过夜培养菌液用煮沸法制DNA模板。应用PCR扩增fimW片段,1.5%凝胶电泳鉴定,目的片段大小为477bp;参考文献合成引物序列如下:fimW-F:5′AACAGTCACTTTGAGCATGGGTT 3′(SEQ ID NO.1);fimW-R:5′GAGTGACTTTGTCTGCTCTTCA 3′(SEQ ID NO.2);反应体系20μL,包含2×Taq Master Mix(Dye Plus)10μL,fimW-F/R(10μM)各1μL,DNA模板2μL,灭菌超纯水6μL补足20μL;PCR反应条件:94℃5min;94℃30s,55℃30s,72℃30s,进行25个循环;72℃10min。结果显示分离株能够扩增出与禽伤寒沙门氏菌标准株U20大小一致的条带(图1)。
将分离株和禽伤寒沙门菌U20菌株单菌落接种于液体LB中37℃过夜震荡培养,进行血清型比较;蔗糖、乳糖、葡萄糖、棉子糖、麦芽糖、甘露醇、靛基质、甘露糖、枸橼酸、卫矛醇、鸟氨酸、赖氨酸、氰化钾、硫化氢、尿素、ONPG、MR试验、V-P试验、半固体琼脂、侧金盏花、硝酸盐还原等微量生化反应比较。
表1表示分离株和禽伤寒沙门氏菌标准株U20的生化特性比较,结果显示两株菌株生化实验结果均一致。
上述结果表明我们分离鉴定到一株沙门氏菌,将其命名为S9。
表1载体菌S9与禽伤寒沙门氏菌标准株U20的生化特性比较
注:“-”表示阴性;“+”表示阳性。
实施例3:惰性载体沙门氏菌S9与鸡源血清无非特异性凝集现象的验证
将惰性载体菌S9转接于LB液体培养基中,振摇过夜后,次日用4℃离心机4000rpm离心l0min,弃上清,用无菌PBS重悬离心洗涤三次后重悬至不同的工作浓度细菌数量梯度。检测前将菌液用涡旋仪混匀,先用无菌PBS/生理盐水和SPF鸡血清进行凝集试验确保菌液无自凝和不出现非特异性凝集现象。在超净台内(20-25℃)取表面洁净普通玻板若干块,用无菌预冷至4℃的PBS将载体菌离心重悬洗涤3次后重悬稀释至规定细菌浓度。用微量移液器吸取一滴约10μL工作浓度的载体菌垂直滴于水平放置的玻板表面上。随后迅速滴加等量的待检血清。用灭菌枪头将菌液与血清充分混合均匀,涂布成直径1-2cm的片状后随即平稳摇动玻板,一般在2min内即可观察试验结果。标准判定状况为室温下2min内,以菌液与待检血清产生絮状或颗粒状肉眼可见沉淀将反应结果判定为阳性,否则判定为阴性。
结果显示,在不同浓度条件下(5-100亿cfu/mL),惰性沙门氏菌载体菌S9都未出现自凝现象,与不同背景多种鸡源血清:SPF鸡血清、鸡细菌性感染阳性血清、鸡寄生虫感染阳性血清、鸡病毒性感染阳性血清和多品种鸡免疫血清,其检测的凝集反应结果都为阴性(表2),说明沙门氏菌S9与鸡源血清不发生非特异性凝集反应,可作为惰性载体菌使用。
表2不同浓度的载体菌S9菌悬液与鸡源血清凝集反应测试结果
注:“-”表示阴性;“+”表示阳性
综上描述了本申请发明的基本原理、惰性载体菌S9的主要鉴别特征和以该惰性载体菌用于间接凝集试验检测方法的开发中提供的一种惰性载体菌和具有的潜在应用前景。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本申请发明的原理,在不脱离本发明功能、原则和范围的前提下,本发明还会不断改进和完善,这些完善都要求在保护本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
序列表
<110> 扬州大学
<120> 一种惰性载体沙门氏菌及其应用
<130> xhx2019052102
<141> 2019-05-21
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
aacagtcact ttgagcatgg gtt 23
<210> 2
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gagtgacttt gtctgctctt ca 22
Claims (8)
1.一种惰性载体沙门氏菌,其特征在于,其菌株代号为S9,保藏在中国普通微生物菌种保藏管理中心(CGMCC),保藏地址为中国北京,保藏编号为CGMCC No.17340,保藏日期为2019年3月18日,分类命名为沙门氏菌Salmonella sp.。
2.根据权利要求1所述的一种惰性载体沙门氏菌,其特征在于,所述的惰性载体沙门氏菌能在LB琼脂或XLD琼脂培养基中培养;其中在LB琼脂培养基中,37℃培养后可形成灰白色圆形菌落;在XLD琼脂培养基中,37℃培养后可形成粉色圆形菌落。
3.根据权利要求1所述的一种惰性载体沙门氏菌,其特征在于,以惰性载体沙门氏菌或其基因组DNA为模板,能通过靶向沙门氏菌种属特异性引物PCR扩增出目的条带,惰性载体沙门氏菌生化特征与标准禽伤寒沙门菌U20株一致。
4.根据权利要求1所述的一种惰性载体沙门氏菌,其特征在于,所述的惰性载体沙门氏菌菌液不存在自凝现象,在工作浓度下的细菌数量与不同遗传背景的多种鸡源血清不发生非特异性凝集反应,鸡源血清包括但不限于SPF鸡血清、健康鸡血清、鸡细菌感染阳性血清、鸡寄生虫感染阳性血清、鸡病毒性感染阳性血清和各种品种鸡免疫血清,其凝集反应结果都为阴性。
5.根据权利要求1所述的惰性载体沙门氏菌,其特征在于:所述的惰性载体沙门氏菌从健康鸡群分离得到。
6.权利要求1所述的惰性载体沙门氏菌在检测抗原或抗体的间接凝集试验中作为惰性载体的应用。
7.权利要求1所述的惰性载体沙门氏菌作为检测抗原或抗体的间接凝集试验用试剂的应用。
8.权利要求1所述的惰性载体沙门氏菌作为鸡相关病原感染检测用试剂的用途。
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910424369.8A CN110218668B (zh) | 2019-05-21 | 2019-05-21 | 一种惰性载体沙门氏菌及其潜在应用 |
US16/963,079 US12085566B2 (en) | 2019-05-21 | 2020-01-13 | Inert carrier Salmonella and potential use thereof |
PCT/CN2020/071626 WO2020233148A1 (zh) | 2019-05-21 | 2020-01-13 | 一种惰性载体沙门氏菌及其潜在应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910424369.8A CN110218668B (zh) | 2019-05-21 | 2019-05-21 | 一种惰性载体沙门氏菌及其潜在应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110218668A true CN110218668A (zh) | 2019-09-10 |
CN110218668B CN110218668B (zh) | 2020-04-03 |
Family
ID=67821442
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910424369.8A Active CN110218668B (zh) | 2019-05-21 | 2019-05-21 | 一种惰性载体沙门氏菌及其潜在应用 |
Country Status (3)
Country | Link |
---|---|
US (1) | US12085566B2 (zh) |
CN (1) | CN110218668B (zh) |
WO (1) | WO2020233148A1 (zh) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111537712A (zh) * | 2020-05-12 | 2020-08-14 | 扬州大学 | 惰性载体间接凝集试验检测系统及其应用 |
CN111560341A (zh) * | 2020-05-19 | 2020-08-21 | 扬州大学 | 一种泛性型惰性载体大肠杆菌及其潜在应用 |
WO2020233148A1 (zh) * | 2019-05-21 | 2020-11-26 | 扬州大学 | 一种惰性载体沙门氏菌及其潜在应用 |
CN112481287A (zh) * | 2020-11-17 | 2021-03-12 | 扬州大学 | 一种表达sef14功能性菌毛的重组菌及其应用 |
WO2021232799A1 (zh) * | 2020-05-19 | 2021-11-25 | 扬州大学 | 一种泛性型惰性载体沙门氏菌及其潜在应用 |
CN116790451A (zh) * | 2023-08-23 | 2023-09-22 | 云南省畜牧兽医科学院 | 鸭源肠炎沙门氏菌抗体检测用抗原、试剂盒及其制备方法 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103837684A (zh) * | 2014-03-27 | 2014-06-04 | 扬州大学 | 快速检测沙门氏菌的抗体试剂及检测方法 |
CN107085103A (zh) * | 2017-05-10 | 2017-08-22 | 中国农业大学 | 一种沙门氏菌抗体乳胶凝集检测方法 |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2316492T3 (es) * | 2000-11-16 | 2009-04-16 | Intervet International Bv | Vacuna contra la salmonella. |
CN101565737A (zh) * | 2008-04-21 | 2009-10-28 | 于维森 | 引起食物中毒沙门氏菌属快检试剂 |
CN107446851B (zh) * | 2017-08-25 | 2020-06-23 | 扬州大学 | 一种鸡白痢凝集抗原及其制备方法 |
CN108570424B (zh) * | 2017-10-13 | 2019-11-15 | 江苏立华牧业股份有限公司 | 一组适合黄羽肉鸡临床应用的鸡白痢沙门氏菌抗原 |
CN110218668B (zh) * | 2019-05-21 | 2020-04-03 | 扬州大学 | 一种惰性载体沙门氏菌及其潜在应用 |
-
2019
- 2019-05-21 CN CN201910424369.8A patent/CN110218668B/zh active Active
-
2020
- 2020-01-13 WO PCT/CN2020/071626 patent/WO2020233148A1/zh active Application Filing
- 2020-01-13 US US16/963,079 patent/US12085566B2/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103837684A (zh) * | 2014-03-27 | 2014-06-04 | 扬州大学 | 快速检测沙门氏菌的抗体试剂及检测方法 |
CN107085103A (zh) * | 2017-05-10 | 2017-08-22 | 中国农业大学 | 一种沙门氏菌抗体乳胶凝集检测方法 |
Non-Patent Citations (2)
Title |
---|
TIMOTHY J.BARRETT: "Improvement of the indirect hemagglutination assay for Salmonella typhi Vi antibodies by use of glutaraldehyde-fixed erythrocytes", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
余辉: "新型凝集载体的研制及其在食品安全快速检测中的初步应用", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020233148A1 (zh) * | 2019-05-21 | 2020-11-26 | 扬州大学 | 一种惰性载体沙门氏菌及其潜在应用 |
CN111537712A (zh) * | 2020-05-12 | 2020-08-14 | 扬州大学 | 惰性载体间接凝集试验检测系统及其应用 |
WO2021227514A1 (zh) * | 2020-05-12 | 2021-11-18 | 扬州大学 | 惰性载体间接凝集试验检测系统及其应用 |
GB2600848A (en) * | 2020-05-12 | 2022-05-11 | Univ Yangzhou | Inert carrier indirect agglutination test detection system and application thereof |
GB2600848B (en) * | 2020-05-12 | 2024-06-26 | Univ Yangzhou | Inert bio-vector indirect agglutination test detection system and uses thereof |
CN111560341A (zh) * | 2020-05-19 | 2020-08-21 | 扬州大学 | 一种泛性型惰性载体大肠杆菌及其潜在应用 |
WO2021232799A1 (zh) * | 2020-05-19 | 2021-11-25 | 扬州大学 | 一种泛性型惰性载体沙门氏菌及其潜在应用 |
WO2021232800A1 (zh) * | 2020-05-19 | 2021-11-25 | 扬州大学 | 一种泛性型惰性载体大肠杆菌及其潜在应用 |
US20230193194A1 (en) * | 2020-05-19 | 2023-06-22 | Yangzhou University | Generic inert bio-vector salmonella sp. and potential uses thereof |
CN112481287A (zh) * | 2020-11-17 | 2021-03-12 | 扬州大学 | 一种表达sef14功能性菌毛的重组菌及其应用 |
CN116790451A (zh) * | 2023-08-23 | 2023-09-22 | 云南省畜牧兽医科学院 | 鸭源肠炎沙门氏菌抗体检测用抗原、试剂盒及其制备方法 |
CN116790451B (zh) * | 2023-08-23 | 2023-11-24 | 云南省畜牧兽医科学院 | 鸭源肠炎沙门氏菌抗体检测用抗原、试剂盒及其制备方法 |
Also Published As
Publication number | Publication date |
---|---|
US20220003763A1 (en) | 2022-01-06 |
US12085566B2 (en) | 2024-09-10 |
CN110218668B (zh) | 2020-04-03 |
WO2020233148A1 (zh) | 2020-11-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110218668A (zh) | 一种惰性载体沙门氏菌及其潜在应用 | |
Gomis et al. | Inclusion body hepatitis as a primary disease in broilers in Saskatchewan, Canada | |
WO2020233147A1 (zh) | 一种惰性载体大肠杆菌及其潜在应用 | |
CN104628833B (zh) | 一种结核感染细胞免疫检测抗原组合物及其应用 | |
CN111537712B (zh) | 惰性载体间接凝集试验检测系统及其应用 | |
RO118885B1 (ro) | Tulpină atenuată, procedeu pentru producerea tulpinii atenuate, vaccin pentru inducerea unui răspuns imun faţă de bacterii lawsonia intracellularis | |
Hopwood et al. | Use of the Pastorex aspergillus antigen latex agglutination test for the diagnosis of invasive aspergillosis. | |
CN104789500A (zh) | 一种鸡白痢染色凝集抗原及其制备方法和应用 | |
CN101879311A (zh) | 猪瘟活疫苗的制备方法及其产品 | |
US20230193194A1 (en) | Generic inert bio-vector salmonella sp. and potential uses thereof | |
CN109486714A (zh) | 一种鸡伤寒沙门氏菌弱毒分离株及其应用 | |
CN112379105A (zh) | 一种prrsv的ipma中和抗体检测方法 | |
CN104628834B (zh) | 一种结核感染t细胞免疫检测抗原及其应用 | |
Krywienczyk et al. | Serological relationship of viruses from some lepidopterous insects | |
CN108842000A (zh) | 用于鉴别DAdV-3和DAdV-A的引物组 | |
CN109709330B (zh) | 一种口蹄疫病毒竞争elisa检测试剂盒 | |
CN104789499A (zh) | 一种鸡白痢琼扩抗原及其制备方法和应用 | |
CN109939225A (zh) | 一株重组鹦鹉热衣原体外膜蛋白momp基因的粗糙型布鲁氏菌及其疫苗生产方法 | |
CN116790451B (zh) | 鸭源肠炎沙门氏菌抗体检测用抗原、试剂盒及其制备方法 | |
RU2268942C1 (ru) | СПОСОБ ВНУТРИВИДОВОЙ ДИФФЕРЕНЦИАЦИИ Vibrio cholerae О139 | |
CN106432487A (zh) | 一种伽氏疏螺旋体多克隆抗体及应用 | |
Hwang et al. | Serologic profiles of chickens infected with subgroup J avian leukosis virus | |
CN112630445A (zh) | 一种csfv的ipma抗体检测方法 | |
Al-Mahmoudi et al. | Rapid Detection of Avian Influenza Virus H9 in broilers by using RT-PCR in Al-Qadisiyah Province | |
JP2681356B2 (ja) | チザー病菌の培養集菌方法 |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
TR01 | Transfer of patent right | ||
TR01 | Transfer of patent right |
Effective date of registration: 20200515 Address after: 242200 Gaohu community, Taozhou Town, Guangde City, Xuancheng City, Anhui Province (within Rongda poultry Co., Ltd.) Patentee after: Anhui Rongda Biotechnology Co.,Ltd. Address before: 225009 No. 88, South University Road, Jiangsu, Yangzhou Patentee before: YANGZHOU University |