CN113881790B - 磁性四氧化三铁@适配体及其与荧光试纸条联合在检测食源性致病菌方面的应用 - Google Patents
磁性四氧化三铁@适配体及其与荧光试纸条联合在检测食源性致病菌方面的应用 Download PDFInfo
- Publication number
- CN113881790B CN113881790B CN202111214519.6A CN202111214519A CN113881790B CN 113881790 B CN113881790 B CN 113881790B CN 202111214519 A CN202111214519 A CN 202111214519A CN 113881790 B CN113881790 B CN 113881790B
- Authority
- CN
- China
- Prior art keywords
- aptamer
- pathogenic bacteria
- food
- test strip
- borne pathogenic
- 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.)
- Active
Links
- 244000052616 bacterial pathogen Species 0.000 title claims abstract description 37
- 238000012360 testing method Methods 0.000 title claims abstract description 31
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 title description 4
- 238000001514 detection method Methods 0.000 claims abstract description 59
- 108091023037 Aptamer Proteins 0.000 claims abstract description 18
- 229910021642 ultra pure water Inorganic materials 0.000 claims abstract description 17
- 239000012498 ultrapure water Substances 0.000 claims abstract description 17
- 238000012408 PCR amplification Methods 0.000 claims abstract description 13
- 238000005406 washing Methods 0.000 claims abstract description 13
- 239000006228 supernatant Substances 0.000 claims abstract description 11
- 239000000843 powder Substances 0.000 claims abstract description 9
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 239000012528 membrane Substances 0.000 claims description 27
- 239000000020 Nitrocellulose Substances 0.000 claims description 23
- 229920001220 nitrocellulos Polymers 0.000 claims description 23
- 238000003908 quality control method Methods 0.000 claims description 22
- 239000000047 product Substances 0.000 claims description 21
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical group N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 20
- 241000186779 Listeria monocytogenes Species 0.000 claims description 19
- 230000001745 anti-biotin effect Effects 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 10
- 229960002685 biotin Drugs 0.000 claims description 10
- 235000020958 biotin Nutrition 0.000 claims description 10
- 239000011616 biotin Substances 0.000 claims description 10
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical group O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 10
- 238000011144 upstream manufacturing Methods 0.000 claims description 8
- 239000012634 fragment Substances 0.000 claims description 6
- 238000010521 absorption reaction Methods 0.000 claims description 5
- 238000009835 boiling Methods 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 239000008055 phosphate buffer solution Substances 0.000 claims description 5
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 4
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 4
- 229940040526 anhydrous sodium acetate Drugs 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 4
- 229940044631 ferric chloride hexahydrate Drugs 0.000 claims description 4
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 229920002678 cellulose Polymers 0.000 claims description 3
- 239000001913 cellulose Substances 0.000 claims description 3
- 229920000728 polyester Polymers 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 2
- 201000010099 disease Diseases 0.000 claims 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 31
- 230000035945 sensitivity Effects 0.000 abstract description 16
- 235000013305 food Nutrition 0.000 abstract description 14
- 238000005516 engineering process Methods 0.000 abstract description 9
- 230000001580 bacterial effect Effects 0.000 description 9
- 239000007788 liquid Substances 0.000 description 7
- 235000015277 pork Nutrition 0.000 description 7
- 230000003321 amplification Effects 0.000 description 6
- 238000001502 gel electrophoresis Methods 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 239000011543 agarose gel Substances 0.000 description 5
- 238000000246 agarose gel electrophoresis Methods 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000010586 diagram Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 241001646719 Escherichia coli O157:H7 Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 238000001157 Fourier transform infrared spectrum Methods 0.000 description 3
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 3
- 241000191967 Staphylococcus aureus Species 0.000 description 3
- 238000003917 TEM image Methods 0.000 description 3
- 241000607272 Vibrio parahaemolyticus Species 0.000 description 3
- 238000002441 X-ray diffraction Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 241000607142 Salmonella Species 0.000 description 2
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 230000001351 cycling effect Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 229910017135 Fe—O Inorganic materials 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 241000186781 Listeria Species 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 240000005319 Sedum acre Species 0.000 description 1
- 235000014327 Sedum acre Nutrition 0.000 description 1
- 241000607598 Vibrio Species 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 244000078673 foodborn pathogen Species 0.000 description 1
- 101150024289 hly gene Proteins 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000004729 solvothermal method Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- BFKJFAAPBSQJPD-UHFFFAOYSA-N tetrafluoroethene Chemical group FC(F)=C(F)F BFKJFAAPBSQJPD-UHFFFAOYSA-N 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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- 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
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- 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
-
- 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
- G01N2333/21—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pseudomonadaceae (F)
-
- 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
- G01N2333/245—Escherichia (G)
-
- 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)
-
- 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
- G01N2333/28—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Vibrionaceae (F)
-
- 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
- G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
- G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种磁性Fe3O4@适配体的制备方法：制备Fe3O4粉末；将Fe3O4粉末用超纯水洗涤后重悬于超纯水中，加入适配体后于室温下震荡20‑30 min，磁分离弃掉上清液，再用超纯水洗涤后重悬于超纯水中，即得。本发明还提供了利用Fe3O4@适配体与荧光试纸条联合在检测食源性致病菌方面的应用。本发明利用PCR扩增技术及荧光信号来提高检测食源性致病菌的灵敏度，具有检测灵敏度高，特异性强，检测时间短等优点，适用于食品样品中致病菌的检测。
Description
技术领域
本发明属于食品安全技术领域，涉及纳米技术和生物技术。具体地，尤其涉及一种磁性四氧化三铁@适配体（Fe3O4@aptamer）以及利用其与荧光试纸条联合在检测食源性致病菌方面的应用，其利用PCR扩增技术及荧光信号来提高检测食源性致病菌的灵敏度。
背景技术
随着食品工业的发展，食品安全问题成为了食品行业以及各界关注的焦点问题之一，其中食源性致病菌的产生是发生食品安全问题的一个重要原因。食源性致病菌常伴随着被污染的食物进入机体,而食物污染可以出现在食物链的任意一个环节。而食源性致病菌检测技术的关键就是要有较高的敏感度、较强的专一性以及尽可能短的分析时间，而现阶段的检测技术不足以对其顺利进行同步、精确及快速的检测，所以很多新型的检测方式已被视为研究的首选。由于纳米材料具有更大的比表面积和特殊的性质，Fe3O4颗粒具有超顺磁性，显示出对外部磁场具有更大的磁化率。
目前，常见的超顺磁Fe3O4纳米颗粒或者Fe3O4磁珠都是带负电荷，将目标识别物（通常为抗体或适配体）通过EDC/NHS偶联的方法与Fe3O4偶联制备捕获剂，这种方法的缺点是：第一偶联效率低且重复性差；第二是羧基的结合有可能会破坏识别物的活性位点。并且目前普通的PCR技术通常通过凝胶电泳法读取结果，这种方法存在耗时、灵敏度低等缺点。针对于此，提出本发明。
发明内容
本发明目的在于克服现有技术缺陷，提供一种磁性Fe3O4@aptamer以及利用其与荧光试纸条联合在检测食源性致病菌方面的应用。本发明利用PCR扩增技术及荧光信号来提高检测食源性致病菌的灵敏度，具有检测灵敏度高，特异性强，检测时间短等优点，特别适用于食品样品中致病菌的检测。
为实现上述目的，本发明采用如下技术方案：
一种磁性Fe3O4@适配体的制备方法，其包括如下步骤：
1）在乙二醇中加入六水氯化铁、无水乙酸钠和EDTA-2Na并混匀，超声20-40 min，转移至反应釜中180-220℃反应8-12h，冷却至室温，经洗涤、干燥，得到Fe3O4粉末；
2）将Fe3O4粉末用超纯水洗涤后重悬于超纯水中，加入适配体后于室温下震荡20-30 min，磁分离弃掉上清液，再用超纯水洗涤后重悬于超纯水中，即得磁性Fe3O4@适配体（带负电荷的适配体通过静电吸附作用结合于正电荷Fe3O4表面）。
具体的，步骤1）中，在60-100 mL乙二醇中加入1-2g六水氯化铁、2-3g无水乙酸钠和0.1-0.2g EDTA-2Na。步骤2）中，Fe3O4和适配体结合时的终浓度分别为1 mg/mL和0.3 μM。
本发明提供了采用上述制备方法制备得到的磁性Fe3O4@适配体。
本发明还提供了上述磁性Fe3O4@适配体与荧光试纸条联合在检测食源性致病菌方面的应用。
上述磁性Fe3O4@适配体与荧光试纸条联合在检测食源性致病菌方面的应用，具体的，包括如下步骤：
a）根据食源性致病菌特异性基因片段设计一对上、下游引物，且上游引物5'端修饰生物素、下游引物5'端修饰荧光素；
b）将食源性致病菌样品与磁性Fe3O4@适配体置于37℃下反应20-40 min，磁吸弃上清，PBS洗涤并重悬于PBS中，100℃煮沸8-15min后冷却至室温，离心，取上清DNA并利用步骤a）中的引物进行PCR扩增，获得PCR产物；具体为：取2 μL上清DNA，步骤a）中的引物各2 μL，25 µL 2× Taq PCR Master Mix buffer，加ddH2O至50 μL进行 PCR反应；PCR混合物首先在95℃预变性5 min，开始循环过程，首先95℃变性30 s、55℃退火30 s、72℃延伸30 s，共进行32个循环。最后，在72°C下继续伸展10分钟即得PCR产物；
c）PCR产物滴加至荧光试纸条上，利用检测仪读取结果。
进一步的，步骤a）中，所述食源性致病菌为单核细胞增生李斯特菌、大肠杆菌O157:H7、沙门氏菌、金黄色葡萄球菌、副溶血性弧菌或铜绿假单胞菌等。步骤b）中，待测的食源性致病菌样品可以是纯培养的食源性致病菌菌液或者含食源性致病菌的食品样品，检测时其与磁性Fe3O4@适配体的体积比为5:1。
进一步的，步骤c）所述荧光试纸条包括底板、以及依次粘接在底板上的样品垫、硝酸纤维素膜和吸水垫；所述硝酸纤维素膜上有检测线和质控线，所述检测线上包被有抗生物素抗体，所述质控线上包被有抗荧光素抗体。
进一步优选，所述样品垫的材质为聚酯纤维素膜，所述吸水垫的材质为吸水滤纸。
具体的，用磷酸缓冲液（10mM，pH 7.4）将抗生物素抗体稀释后，用划膜仪将其包被于硝酸纤维素膜上的检测线；用磷酸缓冲液（10mM，pH 7.4）将抗荧光素抗体稀释后，用划膜仪将其包被于硝酸纤维素膜上的质控线；将包被好的硝酸纤维素膜置于30-45℃条件下干燥1-4 h，获得设有检测线和质控线的硝酸纤维素膜，且检测线上包被有抗生物素抗体、质控线上包被有抗荧光素抗体。
本发明中，实验所用PBS均指浓度0.01 M、pH 7.4的磷酸盐缓冲溶液，并需提前灭菌。
本发明先是制备合成了表面带正电荷的磁性Fe3O4@适配体以捕获和分离致病菌，再利用简单煮沸法提取致病菌DNA并且通过PCR扩增的原理，用于高灵敏检测食源性致病菌。主要包括：先通过溶剂热法合成制备Fe3O4，再将适配体通过正负电荷的静电力吸附作用结合于表面制得Fe3O4@适配体，然后利用适配体标记的磁珠捕获和富集食源性致病菌，通过简单的煮沸法提取DNA，取DNA模板并且引入生物素和荧光素进行PCR扩增；同时制备荧光试纸条，检测线为抗生物素抗体，质控线为抗荧光素抗体；PCR产物的一端有生物素，另一端有荧光素，PCR产物通过试纸条时生物素会被检测线上的抗体捕获，从而检测线也会有荧光信号。本发明是通过正负电荷的静电力吸附食源性致病菌适配体作为食源性致病菌富集/分离载体，通过富集载体进行富集、PCR扩增以及荧光试纸条的荧光信号三个信号放大方式实现检测。本发明检测方法检测灵敏度高，纯培养的菌液的检测限是1×102 CFU/mL；含菌的食品样品的检测限是2×102 CFU/mL；且本方法具有较强的特异性。
本发明利用PCR扩增技术和荧光试纸条，PCR经过32个循环扩增了大量的DNA；荧光试纸条制备方法简单，样品垫、硝酸纤维素膜和吸水垫依次贴合在底板上，其中，抗生物素抗体和抗荧光素抗体分别喷涂于硝酸纤维素膜形成检测线和质控线，具有创新性。本发明具有检测灵敏度高，特异性强，检测时间短等特点。与其它现有的传统检测方法相比，本发明具有如下显著有益效果：
1）检测所用材料与试剂稳定、简单且操作方便快捷；
2）本发明通过食源性致病菌适配体与表面带正电荷磁性Fe3O4的以静电力吸附，效率高；
3）本发明通过磁性载体进行富集、PCR扩增以及荧光试纸条的荧光信号三个信号放大方式确保了本检测方法的灵敏度；
4）本发明对食源性致病菌的选择性包括适配体的识别和特异性引物的扩增，对检测的准确性进行了双重保障；
5）本发明检测快速、灵敏度高、特异性较强，检测时间在4h内，对纯培养的菌液和含菌的食品样品的检测限均低于2×102 CFU/mL。
附图说明
图1为本发明的检测原理示意图；
图2为本发明制备的Fe3O4在不同放大倍数下的TEM图 (A)和粒径分布图(B)；
图3为本发明制备的Fe3O4的XRD图(A)和FT-IR光谱图(B)；
图4为本发明制备的Fe3O4 (A)的ζ电势图和Fe3O4@aptamer (B)的ζ电势图；
图5为本发明中检测不同浓度纯培养的单核细胞增生李斯特菌的荧光试纸条(A，图中从左至右分别代表L. monocytogenes浓度108、107、106、105、104、103、102 CFU/mL以及空白对照)和凝胶电泳图(B，图中1至7分别代表L. monocytogenes浓度1.9×108、2.0×107、1.6×106、1.7×105、1.7×104、2.3×103、1.0×102 CFU/mL，M: RealBand 100 bp DNALadder，C: 空白对照)；
图6为本发明中检测不同浓度单核细胞增生李斯特菌猪肉样品的荧光试纸条(A，图中从左至右分别代表猪肉样品中L. monocytogenes浓度108、107、106、105、104、103、102 CFU/mL以及空白对照)和凝胶电泳图(B，图中1至7分别代表猪肉样品中L. monocytogenes浓度1.3×108、1.4×107、1.3×106、1.2×105、1.3×104、1.3×103、2.0×102 CFU/mL，M:DNA分子量标准 Marker (100~1500 bp)，C: 空白对照)；
图7为本发明中检测单核细胞增生李斯特菌时的检测特异性，即不同食源性致病菌的荧光试纸条可视图(A，图中从左至右依次是：空白对照、单核细胞增生李斯特菌、大肠杆菌O157:H7、鼠伤寒沙门氏菌、金黄色葡萄球菌、副溶血性弧菌、铜绿假单胞菌)和凝胶电泳图(B，图中1至6依次是：单核细胞增生李斯特菌、大肠杆菌O157:H7、鼠伤寒沙门氏菌、金黄色葡萄球菌、副溶血性弧菌、铜绿假单胞菌；M: DNA分子量标准 Marker (100~1500 bp);C: 空白对照)。
具体实施方式
下面结合具体实施例，对本发明做进一步说明。应理解，以下实施例仅用于说明本发明而非用于限制本发明的范围，本领域的技术人员根据上述发明内容作出的一些非本质改进和调整，均在本发明保护范围内。
实施例1
一种磁性Fe3O4@适配体的制备方法，其具体包括如下步骤：
1）在80 mL乙二醇中加入1.633 g六水氯化铁，磁力搅拌8 min；再加入2.4 g无水乙酸钠，磁力搅拌8 min；再加入0.116 g EDTA-2Na，磁力搅拌8 min。超声处理30 min后，将得到的溶液转移至100 mL内衬四氟乙烯的不锈钢高压反应釜中，在200 ℃下加热反应10 h后，冷却至室温。用无水乙醇洗涤3次，室温干燥6 h，得到Fe3O4粉末；
2）取4mg步骤1）制备所得Fe3O4粉末用超纯水洗涤2次，重悬于3988μL超纯水中，加入12μL 100 μM单核细胞增生李斯特菌适配体（序列为5'-TACTATCGCGGAGACAGCGCGGGAGGCACCGGGGA-3'，此产品定制于华大基因，向10 OD适配体粉末中加入290 μL超纯水制得浓度为100 μM的适配体溶液）。于室温下温和摇晃震荡30 min，磁分离弃掉上清液，再用超纯水洗涤2次后重悬于4 mL 超纯水中，获得磁性四氧化三铁@适配体（Fe3O4@aptamer），4℃保存备用。
图2 中A为本发明制备的Fe3O4在不同放大倍数下的TEM图；TEM图中显示：本发明制备的Fe3O4颗粒呈光滑微球。图2中B为本发明制备的Fe3O4的粒径分布图，图中结果显示：Fe3O4颗粒粒径约在300-400 nm之间。
图3中A为本发明制备的Fe3O4的XRD图；XRD图展示出Fe3O4特征峰，对应的晶格面分别为 (220)、 (311)、(400)、(511)、(440)，与Fe3O4的XRD标准卡一致（PDF 26-1136），由XRD图可知本发明制备的Fe3O4是可信的。
图3中B为本发明制备的Fe3O4的FT-IR光谱图；对于Fe3O4的FT-IR光谱图，在1421cm-1和1626 cm-1处的谱图对应于Fe3O4表面羧基，另外在584 cm-1处的峰可归于Fe-O键；由FT-IR光谱图可知本发明制备的Fe3O4是可信的。
图4为本发明制备的Fe3O4和Fe3O4@aptamer的ζ电势图。ζ电势图展示了Fe3O4(A)和Fe3O4@aptamer (B)的ζ电势分别为+32.2±1.2 mV和-25.5±1.2 mV，本发明制备的Fe3O4为正电荷，带负电荷适配体吸附于其表面后形成Fe3O4@aptamer，由电位变化可知适配体已成功吸附到Fe3O4表面。
实施例2
本实施例中，所用到的荧光试纸条经下述方法制备获得：荧光试纸条包括底板、以及依次粘接在底板上的样品垫、硝酸纤维素膜和吸水垫；底板(D70019)、样品垫(F30080)、硝酸纤维素膜(1900703)和吸水垫(H0022)购于上海杰一生物技术有限公司；样品垫宽2cm,硝酸纤维素膜宽2.5cm, 吸水垫宽4cm, 硝酸纤维素膜上有检测线和质控线，检测线和质控线间隔距离0.5cm, 检测线上包被有抗生物素抗体（Anti-Biotin antibody ab53494购于abcam），质控线上包被有抗荧光素抗体（抗荧光素抗体，购于百迈格生物科技有限公司）。样品垫的材质为聚酯纤维素膜，吸水垫的材质为吸水滤纸。用磷酸缓冲液（10mM，pH 7.4）将抗生物素抗体稀释后，用划膜仪将其包被于硝酸纤维素膜上的检测线；用磷酸缓冲液（10mM，pH 7.4）将抗荧光素抗体稀释后，用划膜仪将其包被于硝酸纤维素膜上的质控线；将包被好的硝酸纤维素膜置于30-45℃条件下干燥1-4 h，获得设有检测线和质控线的硝酸纤维素膜，且检测线上包被有抗生物素抗体、质控线上包被有抗荧光素抗体。
上述磁性Fe3O4@适配体与荧光试纸条联合在检测食源性致病菌方面的应用，具体的，包括如下步骤：
a）根据食源性致病菌单核细胞增生李斯特菌（ATCC 15313）特异性基因片段（hly基因）设计一对上、下游引物，此引物扩增出的目标产物片段为375bp。分别为：上游引物LM-hlyF （序列为5'-GTAAGCGGAAAATCTGTCTC-3'）和下游引物LM-hlyR （序列为5'-ATTTCGTTACCTTCAGGATC-3'）；且上游引物5'端修饰生物素、下游引物5'端修饰荧光素（委托华大基因修饰）；
b）将处于对数生长期的单核细胞增生李斯特菌（ATCC 15313）在无菌操作下梯度稀释为108 CFU/mL、107 CFU/mL、106CFU/mL、105 CFU/mL、104 CFU/mL、103 CFU/mL和102 CFU/mL，无菌PBS缓冲液作为对照。将已经稀释好的不同浓度的单核细胞增生李斯特菌菌液与实施例1制备所得Fe3O4@适配体（每个浓度做3组平行）以5:1的体积比置于37 ℃下反应30 min进行捕获。用外置磁铁进行磁吸分离，弃上清后用PBS洗涤1次并重悬于100 μL PBS中，100℃煮沸10min，冷却至室温，离心，取上清液DNA作为模板并利用步骤a）中的引物进行PCR扩增，获得PCR产物；具体为：取2 μL上清DNA，步骤a）中生物素和荧光素标记引物各2 μL，25 µL 2× Taq PCR Master Mix（购于生工生物工程股份有限公司），加ddH2O至50 μL进行PCR扩增。PCR混合物首先在95℃预变性5 min，开始循环过程，首先95℃变性30 s、55℃退火30s、72℃延伸30 s，共进行32个循环。最后，在72℃下继续伸展10分钟即得PCR产物；
c）20 μL PCR产物滴加于荧光试纸条上，1h后利用检测仪读取结果。如图1中B所示，当样品中有目标菌时，PCR可以扩增出产物（PCR产物上同时带有生物素和荧光素），检测线上包被的抗生物素抗体通过捕获PCR产物上的生物素同时具有荧光素的荧光信号，即检测线出现荧光条带；质控线上包被的抗荧光素抗体通过捕获荧光素具有荧光信号，出现荧光条带。当样品中无目标菌时，PCR无法扩增出同时带有生物素和荧光素的产物，因此检测线无荧光条带，只有质控线有荧光条带出现。即阳性结果为质控线、检测线两条荧光条带，阴性结果为仅质控线有荧光条带。
同时利用传统的琼脂糖凝胶法检测PCR产物，通过传统的平板计数法对不同浓度菌液中菌落数进行检测，和本发明作比较，用以说明本发明检测方法具有较高的灵敏度。
结果分析：通过传统平板计数法对不同浓度梯度的单核细胞增生李斯特菌纯菌液进行检测，菌液中实际菌落数为1.9×108、2.0×107、1.6×106、1.7×105、1.7×104、2.3×103、1.0×102 CFU/mL。图5给出了本发明检测不同浓度梯度纯培养的单核细胞增生李斯特菌的荧光试纸条图 (A) 和传统的琼脂糖凝胶电泳图 (B)。图5的A中质控线（C line）和检测线（T line）在不同菌落数（从左至右依次是108、107、106、105、104、103、102 CFU/mL）时都有绿色的荧光条带。PCR产物经2.0% 琼脂糖凝胶电泳40 min，在凝胶成像系统中测定扩增结果，在400bp附近出现目的条带，和预期片段375bp相吻合（见图5中B），表明PCR产物为目标条带，凝胶电泳图（图5中B）验证说明本发明检测方法具有良好的可信度。传统琼脂糖凝胶法对单核细胞增生李斯特菌纯菌液的检测灵敏度为1.6×106 CFU/mL。图5中 A的荧光试纸条可视图显示：本发明对单增李斯特菌的检测限低至1.0×102 CFU/mL，较传统琼脂糖凝胶法更加灵敏，并且与传统的平板计数法（耗时长，需要1-2天）比快速、准确，并且可对不可培养状态的菌进行检测。
实施例3
本发明对猪肉样品中的单核细胞增生李斯特菌进行检测，猪肉样品的处理参考《食品微生物检验》，具体为：取25 g猪里脊肉去离子水冲洗两次，75%的乙醇浸泡10min，紫外杀菌30min，将单核细胞增生李斯特菌菌液注射到肉中，过夜培养，放置225 mL PBS中，拍打震荡得菌悬液并进行梯度稀释。平板计数法获得不同稀释梯度菌液中的实际菌落数为1.3×108、1.4×107、1.3×106、1.2×105、1.3×104、1.3×103、2.0×102 CFU/mL。测定过程参照实施例2。同时利用传统琼脂糖凝胶法检测PCR产物。
检测结果分析：图6给出了本发明检测含不同浓度单核细胞增生李斯特菌猪肉样品的荧光试纸条图 (A)和传统的琼脂糖凝胶凝胶电泳图 (B)。图6的A中质控线（C line）和检测线（T line）在不同菌落数（从左至右依次是108、107、106、105、104、103、102 CFU/mL）时都有绿色的荧光条带。PCR产物经2.0% 琼脂糖凝胶电泳40 min，在凝胶成像系统中测定扩增结果，在400 bp 附近出现目的条带，和预期目标片段相吻合（见图6中B）。传统琼脂糖凝胶法对含不同浓度单核细胞增生李斯特菌猪肉样品的检测灵敏度为1.4×107 CFU/mL（见图6中B）。图6中A的荧光试纸条可视图显示：本发明对单增李斯特菌的检测限低至2.0×102CFU/mL，因此在食品样品里本发明同样具有较好的灵敏度和准确性。
特异性检测：取浓度为107 CFU/mL的单核细胞增生李斯特菌（L. monocytogenes）、大肠杆菌O157:H7（E. coli O157:H7）、鼠伤寒沙门氏菌（S. typhimurium）、金黄色葡萄球菌（S. aureus）、副溶血性弧菌（V . parahemolyticus）和铜绿假单胞菌（P. aeruginosa）参照上述实施例2进行检测，通过凝胶电泳图评价本发明方法的检测特异性。
图7为本发明检测纯培养菌液的检测特异性图；琼脂糖凝胶电泳（图7中B）结果显示只有L. monocytogenes出现目的条带，而其他五种致病菌均没有出现目的条带。本发明荧光试纸条（图7 A）结果显示：只有L. monocytogenes得PCR产物出现阳性结果，其余菌株均没有在检测线出现荧光信号，由此说明本发明检测特异性良好。
以上显示和描述了本发明的基本原理和主要特征以及本发明的优点。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发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。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。
Claims (3)
1.一种磁性Fe3O4@适配体与荧光试纸条联合在检测食源性致病菌方面的应用，所述应用为非疾病诊断目的，其特征在于，包括如下步骤：
a）根据食源性致病菌特异性基因片段设计一对上、下游引物，且上游引物5'端修饰生物素、下游引物5'端修饰荧光素；上游引物LM-hlyF序列为5'-GTAAGCGGAAAATCTGTCTC-3'，下游引物LM-hlyR序列为5'-ATTTCGTTACCTTCAGGATC-3'；
b）将食源性致病菌样品与磁性Fe3O4@适配体置于37℃下反应20-40 min，磁吸弃上清，PBS洗涤并重悬于PBS中，煮沸8-15min后冷却至室温，离心，取上清DNA并利用步骤a）中的引物进行PCR扩增，获得PCR产物；
c）PCR产物滴加至荧光试纸条上，利用检测仪读取结果；
步骤a）中，所述食源性致病菌为单核细胞增生李斯特菌；
步骤b）中，所述磁性Fe3O4@适配体的制备方法包括如下步骤：
1）在乙二醇中加入六水氯化铁、无水乙酸钠和EDTA-2Na并混匀，超声20-40 min，转移至反应釜中180-220℃反应8-12h，冷却至室温，经洗涤、干燥，得到Fe3O4粉末；
2）将Fe3O4粉末用超纯水洗涤后重悬于超纯水中，加入适配体后于室温下震荡20-30min，磁分离弃掉上清液，再用超纯水洗涤后重悬于超纯水中，即得；适配体序列为5'-TACTATCGCGGAGACAGCGCGGGAGGCACCGGGGA-3'；
步骤c）所述荧光试纸条包括底板、以及依次粘接在底板上的样品垫、硝酸纤维素膜和吸水垫；所述硝酸纤维素膜上有检测线和质控线，所述检测线上包被有抗生物素抗体，所述质控线上包被有抗荧光素抗体。
2.如权利要求1所述磁性Fe3O4@适配体与荧光试纸条联合在检测食源性致病菌方面的应用，其特征在于，所述样品垫的材质为聚酯纤维素膜，所述吸水垫的材质为吸水滤纸。
3.如权利要求1所述磁性Fe3O4@适配体与荧光试纸条联合在检测食源性致病菌方面的应用，其特征在于，用磷酸缓冲液将抗生物素抗体稀释后，用划膜仪将其包被于硝酸纤维素膜上的检测线；用磷酸缓冲液将抗荧光素抗体稀释后，用划膜仪将其包被于硝酸纤维素膜上的质控线；将包被好的硝酸纤维素膜置于30-45℃条件下干燥1-4 h。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111214519.6A CN113881790B (zh) | 2021-10-19 | 2021-10-19 | 磁性四氧化三铁@适配体及其与荧光试纸条联合在检测食源性致病菌方面的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111214519.6A CN113881790B (zh) | 2021-10-19 | 2021-10-19 | 磁性四氧化三铁@适配体及其与荧光试纸条联合在检测食源性致病菌方面的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113881790A CN113881790A (zh) | 2022-01-04 |
| CN113881790B true CN113881790B (zh) | 2024-03-19 |
Family
ID=79003424
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111214519.6A Active CN113881790B (zh) | 2021-10-19 | 2021-10-19 | 磁性四氧化三铁@适配体及其与荧光试纸条联合在检测食源性致病菌方面的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113881790B (zh) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113624967B (zh) * | 2021-08-11 | 2024-06-25 | 郑州轻工业大学 | 一种磁性mof@适配体以及利用其检测食源性致病菌的方法 |
| CN114525352B (zh) * | 2022-03-24 | 2024-05-17 | 郑州轻工业大学 | 多重pcr与胶体金试纸条联合高通量检测致病菌的方法 |
| CN117074658B (zh) * | 2023-10-18 | 2023-12-26 | 内蒙古大学 | 一种黄曲霉毒素m1快速检测试剂盒及其检测方法 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103940792A (zh) * | 2014-02-20 | 2014-07-23 | 江南大学 | 一种基于多色上转换荧光标记同时检测三种食源性致病菌的方法 |
| CN105675877A (zh) * | 2016-03-01 | 2016-06-15 | 江南大学 | 一种基于双色时间分辨荧光标记-磁分离适配体识别同时检测两种致病菌的方法 |
| KR20180003818A (ko) * | 2016-07-01 | 2018-01-10 | 원광대학교산학협력단 | 인플루엔자 바이러스에 결합하는 압타머 및 이의 용도 |
| CN107796938A (zh) * | 2017-10-17 | 2018-03-13 | 中国农业科学院农业质量标准与检测技术研究所 | 一种核酸适配体荧光试纸条及其制备方法和应用 |
| CN112881352A (zh) * | 2021-01-07 | 2021-06-01 | 青岛农业大学 | 用于沙门氏菌检测和消杀的核酸适配体-量子点生物传感器、其制备方法及应用 |
-
2021
- 2021-10-19 CN CN202111214519.6A patent/CN113881790B/zh active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103940792A (zh) * | 2014-02-20 | 2014-07-23 | 江南大学 | 一种基于多色上转换荧光标记同时检测三种食源性致病菌的方法 |
| CN105675877A (zh) * | 2016-03-01 | 2016-06-15 | 江南大学 | 一种基于双色时间分辨荧光标记-磁分离适配体识别同时检测两种致病菌的方法 |
| KR20180003818A (ko) * | 2016-07-01 | 2018-01-10 | 원광대학교산학협력단 | 인플루엔자 바이러스에 결합하는 압타머 및 이의 용도 |
| CN107796938A (zh) * | 2017-10-17 | 2018-03-13 | 中国农业科学院农业质量标准与检测技术研究所 | 一种核酸适配体荧光试纸条及其制备方法和应用 |
| CN112881352A (zh) * | 2021-01-07 | 2021-06-01 | 青岛农业大学 | 用于沙门氏菌检测和消杀的核酸适配体-量子点生物传感器、其制备方法及应用 |
Non-Patent Citations (8)
| Title |
|---|
| A gold nanoparticles-assisted multiplex PCR assay for simultaneous detection of Salmonella typhimurium, Listeria monocytogenes and Escherichia coli O157:H7;Juan Du等;《Analytical Methods》;第12卷;第212-217页 * |
| Application of aptamers as molecular recognition elements in lateral flow assays;Ruth Reid等;《Analytical Biochemistry》;第593卷;113574 * |
| Detection of Listeria monocytogenes based on combined aptamers magnetic capture and loop-mediated isothermal amplification;Junli Feng等;《Food Control》;第85卷;第443-452页 * |
| Label-free lateral flow assay for Listeria monocytogenes by aptamer-gated release of signal molecules;Taşbaşı, B. Büşra等;《Analytical Biochemistry》;第587卷;113449 * |
| Multifunctional Fe3O4@Au supraparticle as a promising thermal contrast for an ultrasensitive lateral flow immunoassay;Ting Zhang等;《Talanta》;第222卷;121478 * |
| 基于核酸适配体的功能化磁性纳米粒子的制备及其在样品前处理中的应用研究;甘海娇;《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》(第1期);第31-32页第2.2.5.1节、第2.2.5.2节 * |
| 基于适配体识别的侧向层析法快速检测单核细胞增生李斯特氏菌;卢春霞;李红敏;孙凤霞;康立超;陈亚;任江涛;马朝阳;;食品与生物技术学报(04);第85-92页 * |
| 食源性致病细菌适配体生物传感器研究进展;李雪彤;林英;张园;李颖;吕淑霞;许文涛;;生物技术通报(04);第125-138页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113881790A (zh) | 2022-01-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113881790B (zh) | 磁性四氧化三铁@适配体及其与荧光试纸条联合在检测食源性致病菌方面的应用 | |
| Yang et al. | Magnetic nano-beads based separation combined with propidium monoazide treatment and multiplex PCR assay for simultaneous detection of viable Salmonella Typhimurium, Escherichia coli O157: H7 and Listeria monocytogenes in food products | |
| CN113624967B (zh) | 一种磁性mof@适配体以及利用其检测食源性致病菌的方法 | |
| Chen et al. | Bacteriophage-based nanoprobes for rapid bacteria separation | |
| Bakthavathsalam et al. | Immunomagnetic nanoparticle based quantitative PCR for rapid detection of Salmonella | |
| He et al. | Aptamer-fluorescent silica nanoparticles bioconjugates based dual-color flow cytometry for specific detection of Staphylococcus aureus | |
| Bohara et al. | Surface-modified cobalt ferrite nanoparticles for rapid capture, detection, and removal of pathogens: a potential material for water purification | |
| Huang et al. | A phage-based magnetic relaxation switching biosensor using bioorthogonal reaction signal amplification for salmonella detection in foods | |
| Wu et al. | Nuclear magnetic resonance biosensor based on streptavidin–biotin system and poly-l-lysine macromolecular targeted gadolinium probe for rapid detection of Salmonella in milk | |
| Li et al. | A novel low-field NMR biosensor based on dendritic superparamagnetic iron oxide nanoparticles for the rapid detection of Salmonella in milk | |
| Khosravi et al. | Preparation of immunomagnetic beads coupled with a rhodamine hydrazine immunosensor for the detection of Mycobacterium avium subspecies paratuberculosis in bovine feces, milk, and colostrum | |
| Yue et al. | Rapid detection of Salmonella in milk by a nuclear magnetic resonance biosensor based on the streptavidin-biotin system and O-carboxymethyl chitosan target gadolinium probe | |
| Jin et al. | NMR rapid detection of Salmonella in milk based on ultra-small iron oxide nanobiosensor | |
| Duan et al. | Preparation of immunomagnetic iron-dextran nanoparticles and application in rapid isolation of E. coli O157: H7 from foods | |
| Bai et al. | Portable sensor based on magnetic separation and enzyme-mediated immune nanomaterials for point-of-care testing of Listeria monocytogenes in food | |
| CN114371287A (zh) | 一种基于免疫磁分离和点击化学反应的金黄色葡萄球菌的检测试剂盒及其检测方法 | |
| Dong et al. | A novel rapid detection method for Salmonella based on NMR macromolecular Gd biosensor | |
| CN113984728A (zh) | 一种用于单增李斯特菌快速检测的荧光生物传感器构建方法 | |
| CN113281507A (zh) | 一种金黄色葡萄球菌快速检测方法及试剂盒 | |
| Setterington et al. | Rapid, sensitive, and specific immunomagnetic separation of foodborne pathogens | |
| CN116338171A (zh) | 一种免洗均相检测方法 | |
| CN110501494A (zh) | 一种基于二氧化锰纳米花和荧光材料的微生物检测方法 | |
| CN115010184A (zh) | 一种基于免疫磁珠的金黄色葡萄球菌重组酶聚合酶扩增检测方法 | |
| CN114525352B (zh) | 多重pcr与胶体金试纸条联合高通量检测致病菌的方法 | |
| CN111323596A (zh) | 一种金黄色葡萄球菌检测试剂盒及其制备方法 |
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 |