CN114088484B - 一种萃取粮食与饲料中真菌毒素的方法 - Google Patents
一种萃取粮食与饲料中真菌毒素的方法 Download PDFInfo
- Publication number
- CN114088484B CN114088484B CN202111328045.8A CN202111328045A CN114088484B CN 114088484 B CN114088484 B CN 114088484B CN 202111328045 A CN202111328045 A CN 202111328045A CN 114088484 B CN114088484 B CN 114088484B
- Authority
- CN
- China
- Prior art keywords
- mycotoxin
- extract
- mycotoxins
- extracting
- liquid
- 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
- 231100000678 Mycotoxin Toxicity 0.000 title claims abstract description 58
- 239000002636 mycotoxin Substances 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 31
- 239000007788 liquid Substances 0.000 claims abstract description 23
- MBMQEIFVQACCCH-UHFFFAOYSA-N trans-Zearalenon Natural products O=C1OC(C)CCCC(=O)CCCC=CC2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-UHFFFAOYSA-N 0.000 claims description 32
- MBMQEIFVQACCCH-QBODLPLBSA-N zearalenone Chemical compound O=C1O[C@@H](C)CCCC(=O)CCC\C=C\C2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-QBODLPLBSA-N 0.000 claims description 32
- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 claims description 24
- 229930020125 aflatoxin-B1 Natural products 0.000 claims description 24
- LINOMUASTDIRTM-QGRHZQQGSA-N deoxynivalenol Chemical compound C([C@@]12[C@@]3(C[C@@H](O)[C@H]1O[C@@H]1C=C(C([C@@H](O)[C@@]13CO)=O)C)C)O2 LINOMUASTDIRTM-QGRHZQQGSA-N 0.000 claims description 19
- LINOMUASTDIRTM-UHFFFAOYSA-N vomitoxin hydrate Natural products OCC12C(O)C(=O)C(C)=CC1OC1C(O)CC2(C)C11CO1 LINOMUASTDIRTM-UHFFFAOYSA-N 0.000 claims description 19
- 239000002115 aflatoxin B1 Substances 0.000 claims description 17
- 238000000605 extraction Methods 0.000 claims description 16
- 239000000843 powder Substances 0.000 claims description 9
- 229940051841 polyoxyethylene ether Drugs 0.000 claims description 8
- 229920000056 polyoxyethylene ether Polymers 0.000 claims description 8
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 7
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 7
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 7
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 7
- 239000010414 supernatant solution Substances 0.000 claims description 7
- 239000004094 surface-active agent Substances 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000003018 immunoassay Methods 0.000 claims description 5
- 238000002965 ELISA Methods 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- 150000002191 fatty alcohols Chemical class 0.000 claims description 3
- 238000003317 immunochromatography Methods 0.000 claims description 3
- XSXWOBXNYNULJG-UHFFFAOYSA-N 2-(2,4,4-trimethylpentan-2-yl)phenol Chemical compound CC(C)(C)CC(C)(C)C1=CC=CC=C1O XSXWOBXNYNULJG-UHFFFAOYSA-N 0.000 claims description 2
- 239000002202 Polyethylene glycol Substances 0.000 claims description 2
- -1 ethylphenyl Chemical group 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 238000004445 quantitative analysis Methods 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- 239000003960 organic solvent Substances 0.000 abstract description 7
- 230000008569 process Effects 0.000 abstract description 4
- 231100000331 toxic Toxicity 0.000 abstract description 4
- 230000002588 toxic effect Effects 0.000 abstract description 4
- 239000012895 dilution Substances 0.000 abstract description 3
- 238000010790 dilution Methods 0.000 abstract description 3
- 235000013305 food Nutrition 0.000 abstract description 3
- 238000005119 centrifugation Methods 0.000 abstract description 2
- 230000007547 defect Effects 0.000 abstract description 2
- 238000003912 environmental pollution Methods 0.000 abstract description 2
- 238000001914 filtration Methods 0.000 abstract description 2
- 239000012528 membrane Substances 0.000 description 26
- 239000000243 solution Substances 0.000 description 16
- 239000000427 antigen Substances 0.000 description 11
- 102000036639 antigens Human genes 0.000 description 11
- 108091007433 antigens Proteins 0.000 description 11
- 235000013339 cereals Nutrition 0.000 description 10
- 241000283707 Capra Species 0.000 description 9
- 239000000020 Nitrocellulose Substances 0.000 description 9
- 239000011248 coating agent Substances 0.000 description 9
- 238000000576 coating method Methods 0.000 description 9
- 229920001220 nitrocellulos Polymers 0.000 description 9
- 239000004005 microsphere Substances 0.000 description 8
- 229930195730 Aflatoxin Natural products 0.000 description 7
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 description 7
- 101100449517 Arabidopsis thaliana GRH1 gene Proteins 0.000 description 7
- 101100434479 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) AFB1 gene Proteins 0.000 description 7
- 239000005409 aflatoxin Substances 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 239000003365 glass fiber Substances 0.000 description 5
- 239000003053 toxin Substances 0.000 description 5
- 231100000765 toxin Toxicity 0.000 description 5
- 210000004916 vomit Anatomy 0.000 description 5
- 230000008673 vomiting Effects 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 239000002250 absorbent Substances 0.000 description 3
- 230000002745 absorbent Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000002096 quantum dot Substances 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 235000005822 corn Nutrition 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
- 238000005520 cutting process Methods 0.000 description 2
- 229930002954 deoxynivalenol Natural products 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 206010019851 Hepatotoxicity Diseases 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010029155 Nephropathy toxic Diseases 0.000 description 1
- 208000031320 Teratogenesis Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000002360 explosive Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 239000003008 fumonisin Substances 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007686 hepatotoxicity Effects 0.000 description 1
- 231100000304 hepatotoxicity Toxicity 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000007694 nephrotoxicity Effects 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 229930183344 ochratoxin Natural products 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000006748 scratching Methods 0.000 description 1
- 230000002393 scratching effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种萃取粮食与饲料中真菌毒素的方法,该方法包括:真菌毒素萃取液的制备、真菌毒素的萃取和真菌毒素的检测。本发明中使用的萃取液不含有机溶剂,相对于使用有机溶剂作为萃取液萃取真菌毒素的毒害作用及污染环境等缺点,更绿色环保;本发明的方法省去检测真菌毒素过程中的离心、过滤、稀释等步骤,萃取液与样品混合后可直接检测,减少检测时间,使用更加方便。本发明适用于食品安全检测技术领域。
Description
技术领域
本发明属于食品安全检测技术领域,具体涉及一种萃取粮食与饲料中真菌毒素的方法。
背景技术
粮食是国家的战略物质,是人民的生活必需品,但粮食在生产、加工、运输、贮存的过程中受温度与湿度的影响,会发生霉变而产生有毒有害的真菌毒素。真菌毒素主要包括黄曲霉毒素、伏马毒素、玉米赤霉烯酮、赭曲霉毒素、脱氧雪腐镰刀菌烯醇等。其中多种真菌毒素有致癌、致畸、肝毒性、肾毒性、免疫抑制等等毒害作用,对人与动物有很大的危害。此外,由于许多真菌都能够产生多种真菌毒素化合物,而且许多来源的农产品在整体加工之前可能会聚集在一起,因此在食品和饲料产品中,多种真菌毒素共存的可能性非常高。
目前使用的对粮食饲料中真菌毒素的检测技术主要分为两大类,一类是以国标检测技术为主的大型仪器检测方法,包括高效液相色谱法(HPLC)、液相色谱-质谱法(LC-MS)、气相色谱-质谱法(GC-MS)等;一类是以免疫学分析方法为基础的快速分析技术,如酶联免疫分析法(ELISA)、免疫层析分析(ICA)、时间分辨荧光免疫层析(TRFIA)等。目前样品中真菌毒素的提取大多采用液相萃取技术。液相萃取技术利用相似相溶原理,将目标分子萃取到一种溶剂中,进而实现目标物和杂质的分离。常用的溶剂主要有甲醇、乙腈、二氯甲烷、三氯甲烷、正己烷等有机溶剂,或者由几种溶剂组合而成的复合溶剂。
不同的真菌毒素在不同粮食、不同部位的分布和含量差异很大,且极性各不相同。这就导致在同时测定不同目标、不同基质的多种真菌毒素时,需分别采用不同的萃取液来萃取不同的真菌毒素,且萃取过后要进行不同倍数的稀释,在检测过程中费时费力。且萃取液大多为有机溶剂,对人体健康有害,污染环境。
发明内容
本发明解决的技术问题是提供一种萃取粮食与饲料中真菌毒素的方法,该方法能够实现对粮食、饲料中真菌毒素的高效萃取,萃取液更加环保,萃取过程更加简便快速。
为实现上述目的,本发明第一方面提供如下技术方案:
一种萃取粮食与饲料中真菌毒素的方法,所述方法包括:
S1:真菌毒素萃取液的制备:使用含0.5-2重量%的Tween20、含0.1-1重量%的EDTA的基础缓冲液PBS,将表面活性剂配制成为含0.05-2重量%表面活性剂的缓冲液得到所述真菌毒素萃取液;
S2:真菌毒素的萃取:准确称取1-2g粮食粉末或饲料粉末于烧杯中,加入所述真菌毒素萃取液,搅拌提取2-5分钟,静置后,直接取上清溶液作为待测液;
S3:真菌毒素的检测:通过免疫检测方法定量分析样品中的真菌毒素含量。
作为本发明的一个实施例,步骤S1中,所述基础缓冲液PBS的浓度为5-10mM,pH为7.2-7.4。
作为本发明的一个实施例,步骤S1中,所述表面活性剂包括脂肪醇聚氧乙烯醚类化合物或烷基酚聚氧乙烯醚类化合物,优选为月桂醇聚醚-15、脂肪醇聚氧乙烯醚磷酸酯、烷基酚聚氧乙烯醚磷酸酯、tert-辛基酚聚氧乙烯醚和乙基苯基聚乙二醇中的一种或几种。
作为本发明的一个实施例,步骤S3中,所述通过免疫检测方法定量分析样品中的真菌毒素含量包括:使用侧向流免疫层析、酶联免疫吸附或化学发光免疫分析中的一种或几种免疫检测方法同时定量分析步骤S2中待测液中黄曲霉毒素B1、玉米赤霉烯酮、呕吐毒素的含量。
本发明提供的上述技术方案至少带来的有益效果:
(1)本发明中使用的萃取液不含有机溶剂,相对于使用有机溶剂作为萃取液萃取真菌毒素的毒害作用及污染环境等缺点,更绿色环保;
(2)本发明的方法省去检测真菌毒素过程中的离心、过滤、稀释等步骤,萃取液与样品混合后可直接检测,减少检测时间,使用更加方便;
(3)本发明的方法可以对黄曲霉毒素、玉米赤霉烯酮、脱氧雪腐镰刀菌烯醇三种极性不同的真菌毒素实现同步萃取;
(4)本发明方法使用含有表面活性剂的缓冲液作为萃取液,无使用甲醇、乙醇等易燃易爆有机溶剂面临的运输、储存、管理等成本问题,节约成本。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将对本发明实施方式作进一步地详细描述。
实施例1:玉米中黄曲霉毒素B1(AFB1)、玉米赤霉烯酮(ZEN)、呕吐毒素(DON)的检测
1)取0.17g月桂醇聚醚-15试剂,加入100mL基础缓冲液(50mM,含1重量%Tween20、0.3重量%EDTA),充分溶解制得2mM的月桂醇聚醚-15溶液,作为黄曲霉毒素B1、玉米赤霉烯酮、呕吐毒素三种真菌毒素的萃取液。
2)称取1g玉米样品粉末于50ml烧杯中,备用。
3)将20ml 2mM的月桂醇聚醚-15溶液加入到样品的烧杯中,搅拌2min进行萃取,静置5min后取上清溶液作为待测液。
4)利用EDC/NHS法将黄曲霉毒素单克隆抗体、玉米赤霉烯酮单克隆抗体、呕吐毒素单克隆抗体分别共价偶联在羧基修饰的时间分辨荧光微球上,分别得到时间分辨荧光微球与AFB1单克隆抗体、ZEN单克隆抗体与DON单克隆抗体的偶联物作为免疫微球,并冻干于微孔中,在4℃冰箱保存。利用划膜仪将黄曲霉毒素包被抗原(AFB1-BSA,0.5mg/mL)以及羊抗鼠IgG(1mg/mL)均匀喷涂到硝酸纤维膜(NC膜)上,将玉米赤霉烯酮包被抗原(ZEN-BSA,0.5mg/mL)以及羊抗鼠IgG(1mg/mL)均匀喷涂到硝酸纤维膜(NC膜)上,将呕吐毒素包被抗原(DON-BSA,0.5mg/mL)以及羊抗鼠IgG(1mg/mL)均匀喷涂到硝酸纤维膜(NC膜)上37℃烘干12小时,将玻璃纤维膜、NC膜、吸水纸依次贴在PVC底板上,用切条机切成宽度为4mm的试纸条,分别制成黄曲霉毒素B1、玉米赤霉烯酮、呕吐毒素的时间分辨荧光检测卡。
5)各取100μL待测液直接滴加到三种真菌毒素检测卡上,检测卡静置反应15分钟;
6)最后,通过时间分辨荧光干式定量检测仪器,通过检测卡T线和C线的荧光强度,来确定AFB1、ZEN、DON三种真菌毒素的含量。
实施例2:小麦中黄曲霉毒素B1、玉米赤霉烯酮、呕吐毒素的检测
1)取0.17g月桂醇聚醚-15试剂,加入100mL基础缓冲液(50mM,含1重量%Tween20、0.3重量%EDTA),充分溶解制得2mM的月桂醇聚醚-15溶液,作为黄曲霉毒素B1、玉米赤霉烯酮与呕吐毒素三种真菌毒素的萃取液。
2)称取1g小麦样品粉末于50ml烧杯中,备用。
3)将20ml 2mM的月桂醇聚醚-15溶液加入到样品的烧杯中,搅拌2min进行萃取,静置5min后取上清溶液作为待测液。
4)取平均粒径为25nm的纳米金颗粒的溶液,用0.1mol/L的碳酸钾溶液调节pH值为7.5,向溶液中加入一定浓度的黄曲霉毒素B1单克隆抗体,反应30分钟后,加入封闭缓冲液(20mM磷酸盐缓冲液,pH 7.2,含牛血清白蛋白5重量%、酪蛋白0.3重量%、PVPK12 0.5重量%、海藻酸钠0.02重量%),继续反应30分钟。用喷金划膜仪将其喷到玻璃纤维膜上,37℃烘干24小时,制备黄曲霉毒素检测卡的结合物垫。同样制备玉米赤霉烯酮与呕吐毒素的结合物垫。利用划膜仪分别将黄曲霉毒素包被抗原(AFB1-BSA,0.3mg/mL)以及羊抗鼠IgG(0.5mg/mL)均匀涂到硝酸纤维膜(NC膜)上,37℃烘干4小时,将玉米赤霉烯酮包被抗原(ZEN-BSA,0.5mg/mL)以及羊抗鼠IgG(1mg/mL)均匀喷涂到硝酸纤维膜(NC膜)上,将呕吐毒素包被抗原(DON-BSA,0.5mg/mL)以及羊抗鼠IgG(1mg/mL)均匀喷涂到硝酸纤维膜(NC膜)上37℃烘干12小时,将玻璃纤维膜、结合物垫、NC膜、吸水纸依次贴在PVC底板上,用切条机切成宽度为3.8mm的试纸条。将试纸装载于塑料卡壳中,分别制成黄曲霉毒素B1、玉米赤霉烯酮、呕吐毒素的胶体金检测卡。
5)各取100μL待测液直接滴加到三种真菌毒素检测卡上,检测卡静置反应15分钟;
6)最后,通过手持定量检测仪器读取检测卡T线和C线的数据,来确定AFB1、ZEN、DON三种真菌毒素的含量。
实施例3:大米中黄曲霉毒素B1、玉米赤霉烯酮、呕吐毒素的检测
1)取0.17g月桂醇聚醚-15试剂,加入100mL基础缓冲液(50mM,含1重量%Tween20、0.3重量%EDTA),充分溶解制得2mM的月桂醇聚醚-15溶液,作为黄曲霉毒素B1、玉米赤霉烯酮与呕吐毒素三种真菌毒素的萃取液。
2)称取1g大米样品粉末于50ml烧杯中,备用。
3)将20ml 2mM的月桂醇聚醚-15溶液加入到样品的烧杯中,搅拌2min进行萃取,静置5min后取上清溶液作为待测液。
4)分别将AFB1-BSA、ZEN-BSA、DON-BSA三种真菌毒素的抗原直接固定在不同的微孔板上,洗涤后,分别向包被了三种真菌毒素抗原的微孔中分别加入50μL待测液,后分别加入50μL对应的三种真菌毒素抗体溶液于37℃孵育,1h后洗涤,加入100μL辣根过氧化物酶标记二抗于37℃孵育1h,洗涤后加入显色液显色,37℃孵育15min后加入终止液终止反应,利用酶标仪测定各孔OD值,计算样品中的AFB1、ZEN、DON三种真菌毒素的含量。
实施例4:全价配合饲料中黄曲霉毒素B1、玉米赤霉烯酮、呕吐毒素的检测
1)取0.17g月桂醇聚醚-15试剂,加入100mL基础缓冲液(50mM,含1重量%Tween20、0.3重量%EDTA),充分溶解制得2mM的月桂醇聚醚-15溶液,作为黄曲霉毒素B1、玉米赤霉烯酮与呕吐毒素三种真菌毒素的萃取液。
2)称取1g全价配合饲料样品粉末于50ml烧杯中,备用。
3)将20ml 2mM的月桂醇聚醚-15溶液加入到样品的烧杯中,搅拌2min进行萃取,静置5min后取上清溶液作为待测液。
4)分别将黄曲霉毒素B1、玉米赤霉烯酮、呕吐毒素三种真菌毒素的单克隆抗体固定到96孔板的不同微孔中,洗涤后,分别向含有三种真菌毒素抗体的微孔中加入100μL待测液,37℃孵育1h,洗涤后加入100μL酶标抗体于37℃孵育1h,洗涤后加入100μL酶底物进行显色,经酶标仪读取各微孔的吸光度值计算样品中的AFB1、ZEN、DON三种真菌毒素的含量。
实施例5:混合饲料中黄曲霉毒素B1、玉米赤霉烯酮、呕吐毒素的检测
1)取0.17g月桂醇聚醚-15试剂,加入100mL基础缓冲液(50mM,含1重量%Tween20、0.3重量%EDTA),充分溶解制得2mM的月桂醇聚醚-15溶液,作为黄曲霉毒素B1、玉米赤霉烯酮与呕吐毒素三种真菌毒素的萃取液。
2)称取1g混合饲料样品粉末于50ml烧杯中,备用。
3)将20ml 2mM的月桂醇聚醚-15溶液加入到样品的烧杯中,搅拌2min进行萃取,静置5min后取上清溶液作为待测液。
4)利用EDC/NHS法将黄曲霉毒素单克隆抗体、玉米赤霉烯酮单克隆抗体、呕吐毒素单克隆抗体分别共价偶联在羧基修饰的量子点上,分别得到量子点与AFB1单克隆抗体、ZEN单克隆抗体与DON单克隆抗体的偶联物作为免疫微球,并冻干于微孔中,在4℃冰箱保存。利用划膜仪将黄曲霉毒素包被抗原(AFB1-BSA,0.5mg/mL)以及羊抗鼠IgG(1mg/mL)均匀喷涂到硝酸纤维膜(NC膜)上,将玉米赤霉烯酮包被抗原(ZEN-BSA,0.5mg/mL)以及羊抗鼠IgG(1mg/mL)均匀喷涂到硝酸纤维膜(NC膜)上,将呕吐毒素包被抗原(DON-BSA,0.5mg/mL)以及羊抗鼠IgG(1mg/mL)均匀喷涂到硝酸纤维膜(NC膜)上37℃烘干12小时,将玻璃纤维膜、NC膜、吸水纸依次贴在PVC底板上,用切条机切成宽度为4mm的试纸条,分别制成黄曲霉毒素B1、玉米赤霉烯酮、呕吐毒素的量子点检测卡。
5)各取100μL待测液直接滴加到三种真菌毒素检测卡上,检测卡静置反应15分钟;
6)最后,通过荧光定量检测仪器,通过检测卡T线和C线的荧光强度,来确定AFB1、ZEN、DON三种真菌毒素的含量。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (1)
1.一种萃取粮食与饲料中真菌毒素的方法,其特征在于,所述方法包括:
S1:真菌毒素萃取液的制备:使用含0.5-2重量%的Tween20、含0.1-1重量%的EDTA的基础缓冲液PBS,将表面活性剂配制成为含0.05-2重量%表面活性剂的缓冲液得到所述真菌毒素萃取液;
所述基础缓冲液PBS的浓度为5-10mM,pH为7.2-7.4;
所述表面活性剂为月桂醇聚醚-15、脂肪醇聚氧乙烯醚磷酸酯、烷基酚聚氧乙烯醚磷酸酯、tert-辛基酚聚氧乙烯醚和乙基苯基聚乙二醇中的一种或几种;
S2:真菌毒素的萃取:准确称取1-2g粮食粉末或饲料粉末于烧杯中,加入所述真菌毒素萃取液,搅拌提取2-5分钟,静置后,直接取上清溶液作为待测液;
S3:真菌毒素的检测:通过免疫检测方法定量分析样品中的真菌毒素含量;
所述通过免疫检测方法定量分析样品中的真菌毒素含量包括:使用侧向流免疫层析、酶联免疫吸附或化学发光免疫分析中的一种或几种免疫检测方法同时定量分析步骤S2中待测液中黄曲霉毒素B1、玉米赤霉烯酮、呕吐毒素的含量。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111328045.8A CN114088484B (zh) | 2021-11-10 | 2021-11-10 | 一种萃取粮食与饲料中真菌毒素的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111328045.8A CN114088484B (zh) | 2021-11-10 | 2021-11-10 | 一种萃取粮食与饲料中真菌毒素的方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114088484A CN114088484A (zh) | 2022-02-25 |
CN114088484B true CN114088484B (zh) | 2024-07-05 |
Family
ID=80299699
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111328045.8A Active CN114088484B (zh) | 2021-11-10 | 2021-11-10 | 一种萃取粮食与饲料中真菌毒素的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114088484B (zh) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112462053A (zh) * | 2020-11-27 | 2021-03-09 | 山东省粮油检测中心(山东省救灾物资储备管理中心) | 一种能同时检测三种真菌毒素的荧光定量试纸条及其制备方法和应用 |
Family Cites Families (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP3739319B2 (ja) * | 1999-09-07 | 2006-01-25 | 協和メデックス株式会社 | トリコテセン系マイコトキシンの検出方法及びその試薬 |
US20030073249A1 (en) * | 2001-07-07 | 2003-04-17 | Lee Duen | Allergen detection chip |
WO2003029479A2 (en) * | 2001-08-10 | 2003-04-10 | Regents Of The University Of California | Sensitive and rapid detection of pathogenic organisms and toxins using fluorescent polymeric lipids |
CA2526691A1 (en) * | 2003-05-23 | 2005-04-28 | Board Of Regents - The University Of Texas System | High throughput screening of aptamer libraries for specific binding to proteins on viruses and other pathogens |
CN101949854B (zh) * | 2010-09-14 | 2012-10-17 | 云南健牛生物科技有限公司 | 一种快速检测黄曲霉毒素含量的方法 |
WO2013116847A1 (en) * | 2012-02-03 | 2013-08-08 | Charm Sciences, Inc. | Extraction of mycotoxins |
CN103604902B (zh) * | 2013-12-06 | 2016-02-17 | 张淑金 | 谷物和食品中快速检测黄曲霉毒素含量的方法 |
CN104749356B (zh) * | 2015-01-07 | 2017-08-08 | 中国检验检疫科学研究院 | 呕吐毒素的均相免疫检测试剂盒及检测方法 |
CN104749009B (zh) * | 2015-03-30 | 2018-05-04 | 上海云泽生物科技有限公司 | 用于免疫分析的免疫抑制剂药物提取试剂 |
CN105424923B (zh) * | 2015-09-10 | 2018-01-05 | 南京微测生物科技有限公司 | 彩色‑荧光双功能免疫层析试纸条及其制备方法 |
CN206832820U (zh) * | 2017-06-19 | 2018-01-02 | 武汉华美生物工程有限公司 | 一种检测包含黄曲霉毒素的真菌毒素用免疫层析试纸条 |
CN107632091B (zh) * | 2017-09-14 | 2020-04-10 | 南京财经大学 | 一种同时检测小麦中多种真菌毒素的方法 |
CN113125725A (zh) * | 2019-12-30 | 2021-07-16 | 江苏维赛科技生物发展有限公司 | 一种同时检测黄曲霉毒素b1和玉米赤霉烯酮的检测卡 |
-
2021
- 2021-11-10 CN CN202111328045.8A patent/CN114088484B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112462053A (zh) * | 2020-11-27 | 2021-03-09 | 山东省粮油检测中心(山东省救灾物资储备管理中心) | 一种能同时检测三种真菌毒素的荧光定量试纸条及其制备方法和应用 |
Also Published As
Publication number | Publication date |
---|---|
CN114088484A (zh) | 2022-02-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Wang et al. | Application of suspension array for simultaneous detection of four different mycotoxins in corn and peanut | |
Krska et al. | Analysis of Fusarium toxins in feed | |
CN103018458B (zh) | 超灵敏黄曲霉毒素b1酶联免疫检测试剂盒 | |
Sun et al. | One-step time-resolved fluorescence microsphere immunochromatographic test strip for quantitative and simultaneous detection of DON and ZEN | |
Zhang et al. | Dual flow immunochromatographic assay for rapid and simultaneous quantitative detection of ochratoxin A and zearalenone in corn, wheat, and feed samples | |
Sun et al. | Novel dual immunochromatographic test strip based on double antibodies and biotin-streptavidin system for simultaneous sensitive detection of aflatoxin M1 and ochratoxin A in milk | |
CN103163297A (zh) | 多功能荧光免疫层析快速定量检测卡 | |
CN102955031A (zh) | 检测黄曲霉毒素b1药物的酶联免疫试剂盒及其应用 | |
CN106324243A (zh) | 一种胶体金免疫层析试纸条及其制备和使用方法 | |
CN104076154A (zh) | 检测叶酸的酶联免疫试剂盒及其应用 | |
CN109061134B (zh) | 检测沙丁胺醇的荧光微球-胶体金双显色定性定量免疫层析试纸条及其制备方法 | |
CN111089956A (zh) | 三重定量检测镰刀菌毒素的荧光微球免疫层析试纸条、其制备方法和应用 | |
Li et al. | An immunochromatographic assay for rapid and direct detection of 3‐amino‐5‐morpholino‐2‐oxazolidone (AMOZ) in meat and feed samples | |
CN102072958A (zh) | 检测黄曲霉毒素b1的化学发光免疫分析测定试剂盒及其制备、使用方法 | |
Anfossi et al. | Lateral flow immunoassays for aflatoxins B and G and for aflatoxin M1 | |
CN106198966A (zh) | 一种检测黄曲霉毒素的近红外荧光免疫层析试剂盒及其应用 | |
CN114088484B (zh) | 一种萃取粮食与饲料中真菌毒素的方法 | |
CN104749356B (zh) | 呕吐毒素的均相免疫检测试剂盒及检测方法 | |
Chen et al. | Well-oriented immobilized immunoaffinity magnetic beads for detection of fumonisins in grains and feeds via pre-column automatic derivatization of high-performance liquid chromatography | |
Yakubu et al. | Aflatoxin: Occurrence, regulation, and detection in food and feed | |
Sheng et al. | Quantitative determination of four mycotoxins in cereal by fluorescent microsphere based immunochromatographic assay | |
WO2023207166A1 (zh) | 一种同时检测谷物中多种真菌毒素和重金属Cd的五联荧光免疫定量试纸条 | |
Becheva et al. | Aflatoxin B1 determination in peanuts by magnetic nanoparticle–based immunofluorescence assay | |
Pestka | Application of immunology to the analysis and toxicity assessment of mycotoxins | |
Pestka | High performance thin layer chromatography ELISAGRAM: Application of a multi-hapten immunoassay to analysis of the zearalenone and aflatoxin mycotoxin families |
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 |