CN106770090B - 一种快速检测黄曲霉毒素m1的方法 - Google Patents
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- MJBWDEQAUQTVKK-IAGOWNOFSA-N aflatoxin M1 Chemical compound C=1([C@]2(O)C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O MJBWDEQAUQTVKK-IAGOWNOFSA-N 0.000 title claims abstract description 48
- 229930073161 aflatoxin M1 Natural products 0.000 title claims abstract description 41
- 239000002108 aflatoxin M1 Substances 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 18
- 150000004032 porphyrins Chemical class 0.000 claims abstract description 21
- 239000000975 dye Substances 0.000 claims abstract description 17
- 238000001514 detection method Methods 0.000 claims abstract description 16
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 7
- 230000005284 excitation Effects 0.000 claims description 6
- 229930195730 Aflatoxin Natural products 0.000 description 6
- 239000005409 aflatoxin Substances 0.000 description 6
- 239000003053 toxin Substances 0.000 description 6
- 231100000765 toxin Toxicity 0.000 description 6
- 108700012359 toxins Proteins 0.000 description 6
- 235000013336 milk Nutrition 0.000 description 5
- 239000008267 milk Substances 0.000 description 5
- 210000004080 milk Anatomy 0.000 description 5
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 description 4
- MBMQEIFVQACCCH-UHFFFAOYSA-N trans-Zearalenon Natural products O=C1OC(C)CCCC(=O)CCCC=CC2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-UHFFFAOYSA-N 0.000 description 4
- 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 description 4
- 235000013305 food Nutrition 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- BXFOFFBJRFZBQZ-QYWOHJEZSA-N T-2 toxin Chemical compound C([C@@]12[C@]3(C)[C@H](OC(C)=O)[C@@H](O)[C@H]1O[C@H]1[C@]3(COC(C)=O)C[C@@H](C(=C1)C)OC(=O)CC(C)C)O2 BXFOFFBJRFZBQZ-QYWOHJEZSA-N 0.000 description 2
- 239000002115 aflatoxin B1 Substances 0.000 description 2
- 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 description 2
- 229930020125 aflatoxin-B1 Natural products 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 235000013365 dairy product Nutrition 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- RWQKHEORZBHNRI-BMIGLBTASA-N ochratoxin A Chemical compound C([C@H](NC(=O)C1=CC(Cl)=C2C[C@H](OC(=O)C2=C1O)C)C(O)=O)C1=CC=CC=C1 RWQKHEORZBHNRI-BMIGLBTASA-N 0.000 description 2
- 235000017060 Arachis glabrata Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000018262 Arachis monticola Nutrition 0.000 description 1
- 241000228197 Aspergillus flavus Species 0.000 description 1
- 241000228230 Aspergillus parasiticus Species 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000007059 acute toxicity Effects 0.000 description 1
- 231100000403 acute toxicity Toxicity 0.000 description 1
- 235000008452 baby food Nutrition 0.000 description 1
- 231100000357 carcinogen Toxicity 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007665 chronic toxicity Effects 0.000 description 1
- 231100000160 chronic toxicity Toxicity 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
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- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
本发明公开了一种快速检测黄曲霉毒素M1的新方法，主要是基于非标记卟啉荧光染料检测黄曲霉毒素M1。该方法是将黄曲霉毒素M1与卟啉荧光染料混合后，染料的荧光强度增加，通过检测荧光强度来实现对黄曲霉毒素M1的快速、定量检测。本发明提供的方法简单、特异性强、检测速度快，能够满足现场快速、准确的检测要求。
Description
技术领域
本发明属于检测技术领域，具体是涉及一种基于非标记卟啉染料检测黄曲霉毒素M1的快速检测方法。
背景技术
黄曲霉毒素为二氢呋喃香豆素的衍生物，是由黄曲霉和寄生曲霉产生的一种次级代谢产物，具有急性毒性、慢性毒性和致癌性。黄曲霉毒素常存在于玉米、花生、谷物等食品中，被世界卫生组织划定为1类致癌物，世界各国对黄曲霉毒素都制定了严格的限量标准。牛乳及乳制品是易受黄曲霉毒素污染的食品之一。当奶牛摄入被黄曲霉毒素污染的饲料后，黄曲霉毒素在其体内可被羟化而产生黄曲霉毒素M1(Aflatoxin M1,AFM1)，所产的牛奶中就含有AFM1。人们食用该牛奶后，会对人体的健康造成严重的危害。欧盟国家规定牛奶中AFM1含量不能超过0.5μg/kg，婴儿食品中不能超过0.025μg/kg。我国规定乳及乳制品中AFM1的限量为0.5μg/kg。建立快速、高灵敏度AFM1的检测方法对于食品安全检测及保护国民身体健康具有重要意义。
目前，国内外检测AFM1的方法主要有色谱法和酶联免疫法。色谱法具有灵敏度高、检测结果好等特点，但是样品前处理过程较为复杂、耗时，并且需要昂贵的大型仪器，难以满足现场快速检测。免疫法是一种常用的检测方法，具有快速、灵敏等特点，但是需要使用价格昂贵且易失活的酶、抗体等生物试剂。因此，需要开发一种简单灵敏的AFM1的快速检测方法。
发明内容
有鉴于此，本发明创造旨在提出一种快速检测黄曲霉毒素M1的新方法。为达到上述目的，本发明创造的技术方案如下：
提供一种快速检测黄曲霉毒素M1的新方法，黄曲霉毒素M1与卟啉染料相互作用后，增强卟啉染料的荧光。通过检测荧光强度实现对黄曲霉毒素M1的快速检测。
本发明具体包括以下步骤：
(1)黄曲霉毒素M1溶液中加入2μM卟啉染料，用荧光分光光度仪检测卟啉染料的荧光强度，激发波长为399nm，发射波长为619nm；
(2)根据黄曲霉毒素M1的浓度与卟啉荧光染料的荧光强度拟合曲线。
优选的，所述黄曲霉毒素M1浓度为0.01-2ng/mL。
优选的，所述的卟啉染料是NMM或PPIX中的一种。
相对于现有技术，本发明创造所述的黄曲霉毒素M1的检测方法具有以下优势：
本发明的优点是：本发明利用非标记卟啉荧光染料直接与黄曲霉毒素M1作用，具有较高的灵敏度和特异性，同时缩短了检测时间，仅需几分钟。
附图说明
构成本发明创造的一部分的附图用来提供对本发明创造的进一步理解，本发明创造的示意性实施例及其说明用于解释本发明创造，并不构成对本发明创造的不当限定。在附图中：
图1为本发明创造实施例所述的黄曲霉毒素M1在激发波长399nm和
365nm的荧光光谱图。
图2为本发明创造实施例所述的黄曲霉毒素M1与卟啉染料NMM的荧
光强度拟合曲线。
图3为本发明创造实施例所述的卟啉染料NMM的特异性实验数据。
图4为卟啉染料PPIX的特异性实验数据。
具体实施方式
为了使本发明上述特征和优点更加清楚和容易理解，下面将结合附图对本发明的实施方式作进一步详细描述。
实施例1
黄曲霉毒素M1的快速检测方法
用荧光分光光度计测10ng/mL黄曲霉毒素M1的荧光强度(如图1所示)，激发波长为365nm和399nm。从图中可以看出，黄曲霉毒素M1在619nm处没有峰。0.01-2ng/mL的黄曲霉毒素M1标准液与2μM卟啉荧光染料NMM混合后，利用荧光分光光度仪测其荧光强度。激发波长为399nm，发射波长为619nm。以黄曲霉毒素M1浓度为横坐标，以(F-F0)/(F'-F0)为纵坐标做拟合曲线，其中F0、F和F’分别表示没有黄曲霉毒素M1时NMM的荧光强度，添加不同浓度黄曲霉毒素M1后NMM的荧光强度及加入2ng/mL黄曲霉毒素M1时NMM的荧光强度(如图2所示)。
实施例2
选择黄曲霉毒素B1(AFB1)、展青梅毒素(PAT)、玉米赤霉烯酮(ZEN)、赭曲霉毒素A(OTA)和T-2毒素作为竞争物，研究该方法的特异性。配制浓度为1ng/mL黄曲霉毒素M1和浓度为10ng/mL竞争毒素。分别与卟啉染料NMM混合，利用荧光分光光度仪测其荧光值。如图3所示，黄曲霉毒素M1可以强烈的增加NMM的荧光。而竞争毒素对其荧光影响较小。说明该方法对黄曲霉毒素M1具有特异性。
实施例3
选择黄曲霉毒素B1(AFB1)、展青梅毒素(PAT)、玉米赤霉烯酮(ZEN)、赭曲霉毒素A(OTA)和T-2毒素作为竞争物，研究以PPIX为非荧光标记卟啉染料时该方法的特异性。配制浓度为1ng/mL黄曲霉毒素M1和浓度为10ng/mL竞争毒素。分别与卟啉染料PPIX混合，利用荧光分光光度仪测其荧光值。激发波长为410nm，发射波长为624nm。如图4所示，黄曲霉毒素M1可以强烈的增加PPIX的荧光。而竞争毒素对其荧光影响较小。说明该方法对黄曲霉毒素M1具有特异性。
以上所述仅为本发明创造的较佳实施例而已，并不用于限制本发明创造，凡在本发明创造的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明创造的保护范围之内。
Claims (2)
1.一种快速检测黄曲霉毒素M1的方法,将黄曲霉毒素M1直接与卟啉染料混合,通过检测染料荧光强度来实现对黄曲霉毒素M1的定量检测,其特征在于,包括如下步骤:
(1)黄曲霉毒素M1溶液中加入2μM卟啉染料,用荧光分光光度仪检测卟啉染料的荧光强度,激发波长为399nm,发射波长为619nm; 所述的卟啉染料是NMM或PPIX中的一种；
(2)根据黄曲霉毒素M1的浓度与卟啉荧光染料的荧光强度拟合曲线。
2.根据权利要求1所述的一种快速检测黄曲霉毒素M1的方法,其特征在于,所述黄曲霉毒素M1浓度为0.01-2ng/mL。
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| CN103217528A (zh) * | 2012-11-30 | 2013-07-24 | 中国农业科学院油料作物研究所 | 一种检测黄曲霉毒素b1含量的非标记免疫分析方法 |
| CN104897863A (zh) * | 2014-03-06 | 2015-09-09 | 北京勤邦生物技术有限公司 | 一种检测黄曲霉毒素m1的荧光微球免疫层析试纸条及方法 |
| CN105486672A (zh) * | 2016-01-20 | 2016-04-13 | 西南大学 | Afm1荧光增敏剂 |
| CN105784990A (zh) * | 2016-05-17 | 2016-07-20 | 中国农业科学院农业质量标准与检测技术研究所 | 一种利用适配体检测黄曲霉毒素b1或m1的试纸条 |
| CN105866416A (zh) * | 2016-05-18 | 2016-08-17 | 深圳市正海生物科技有限公司 | 免疫层析试纸条、便携式检测仪及检测方法 |
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| CN103217528A (zh) * | 2012-11-30 | 2013-07-24 | 中国农业科学院油料作物研究所 | 一种检测黄曲霉毒素b1含量的非标记免疫分析方法 |
| CN104897863A (zh) * | 2014-03-06 | 2015-09-09 | 北京勤邦生物技术有限公司 | 一种检测黄曲霉毒素m1的荧光微球免疫层析试纸条及方法 |
| CN105486672A (zh) * | 2016-01-20 | 2016-04-13 | 西南大学 | Afm1荧光增敏剂 |
| CN105784990A (zh) * | 2016-05-17 | 2016-07-20 | 中国农业科学院农业质量标准与检测技术研究所 | 一种利用适配体检测黄曲霉毒素b1或m1的试纸条 |
| CN105866416A (zh) * | 2016-05-18 | 2016-08-17 | 深圳市正海生物科技有限公司 | 免疫层析试纸条、便携式检测仪及检测方法 |
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