高灵敏黄曲霉毒素B1免疫层析定量检测法的建立

以胶体金为标记物,借助于胶体金读取仪,建立了高灵敏的定量检测黄曲霉毒素B_1的胶体金免疫层析方法。试纸条制备时,当标记溶液pH为6.0,检测线全抗原的使用浓度为1.5mg/mL,金标抗体浓度为4μg/mL、用量为1.2μL时,所建立方法的灵敏度为5.7ng/L。通过加速保存试验,结果显示该试纸条的保存期大于1年。该试纸条应用于实际谷物及酱油加标样本检测,证明该方法可满足食品快速检测的需要。     

胶体金免疫层析法快速检测黄曲霉毒素B1的研究

本文应用胶体金免疫层析技术,建立了一种快速检测食品中黄曲霉毒素B1的方法。采用柠檬酸三钠还原法制备胶体金颗粒,标记抗黄曲霉毒素B1单克隆抗体并喷于玻璃纤维上,黄曲霉毒素B1偶联抗原和二抗鼠抗驴分别结合于硝酸纤维膜上,依次将样本垫、胶金垫、硝酸纤维膜和吸水纸组装切割成胶体金试纸条并装入检测卡中。测试结果表明黄曲霉毒素B1快速检测试纸条的灵敏度为5ng/ml,检测时间为10min,批内和批间重复性为100%,假阳性率和假阴性率均为0。使用简单方便,非常适合现场快速检测黄曲霉毒素B1。     

黄曲霉毒素B1的检测方法

随着农业市场的不断发展, 伴随产生了一系列的食品安全质量问题, 尤其是毒性最强的黄曲霉毒素B1所引起的安全问题。粮食安全问题是一个不容小觑的重大问题, 现在也越来越受到老百姓的重视。本文介绍了黄曲霉毒素B1(aflatoxin B1, AFB1)及其毒性危害; 综述了几种黄曲霉毒素B1的检测方法, 分别包括: 薄层层析法(thin–layer chromatography, TLC)、高效液相色谱法(high performance liquid chromatography, HPLC)、酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)、金标试纸法(gold labeled immunochromatographic assay, GICA)等, 并对这几种方法进行了分析和比较, 以期为黄曲霉毒素B1的监测提供参考。     

时间分辨荧光免疫层析试纸条在油料饼粕黄曲霉毒素B_1检测中的应用

采用黄曲霉毒素时间分辨荧光免疫层析试纸条及配套的时间分辨荧光速测仪,对油料饼粕中黄曲霉毒素B1的快速检测进行了应用研究。该时间分辨荧光免疫分析技术是基于时间分辨荧光免疫层析试纸条和载有Eu（Ⅲ）标记特异性单克隆抗体的样品瓶建立的检测技术。时间分辨荧光速测仪可内置标准曲线,直接输出检测结果。对6种油料饼粕做黄曲霉毒素B1添加回收率实验,回收率在70%~120%之间,批间、批内变异系数〈15%。在实际样品的检测中,时间分辨荧光免疫层析试纸条检测技术与液相色谱-串联质谱法相比,检测结果相对误差〈15%。时间分辨荧光免疫层析试纸条检测技术测定快速、准确,技术稳定、可靠,设备经济、小型,适用于大批量油料饼粕样品的快速检测和风险评估。     

胶体金免疫层析技术快速检测谷物中3种真菌毒素的研究

目的应用胶体金免疫层析技术开发一种快速检测谷物中黄曲霉毒素B1(AFB1)、玉米赤霉烯酮(ZEN)和呕吐毒素(DON)的三联检测卡。方法优化胶体金溶液p H值、金标抗体量及抗原包被量制备3种胶体金试纸条,将试纸条组装成检测卡,研究该卡的检测限、准确度、特异性和稳定性。结果 AFB_1、ZEN和DON 3种试纸条的胶体金溶液最适p H值分别为7.5、7.5和7.0,金标抗体量分别为4.2、7.2和9.6μg/ml,抗原包被量分别为1.0、1.4和0.8 mg/ml。检测卡的检测结果与仪器检测结果基本一致,重复性较好,与结构类似物及其他霉菌毒素无交叉反应,在室温条件下可以保存12个月。结论该三联检测卡操作简便、快速、准确、稳定,且能同时检测3种真菌毒素,在谷物的现场检测中有较好的应用前景。     

氯化钙对花生中黄曲霉毒素B1提取及其快速检测结果的影响

以氯化钙为蛋白沉淀剂、黄曲霉毒素B1为靶标,探究了70％甲醇水（V/V）提取液中不同含量氯化钙对花生中黄曲霉毒素提取及其免疫检测结果的影响;采用样品加标回收的方法,研究比较了五种不同含量氯化钙对胶体金免疫层析试纸条和ELISA检测结果的影响。研究结果表明,样品经含1％CaCl2（m／V）的甲醇水处理后,用胶体金免疫层析试纸条检测时,试纸条T线颜色梯度变化,不同浓度加标回收率等参数均得到明显改善,前处理效果显著;选取20份花生实际样品,经过这种前处理之后,进行胶体金免疫层析试纸条和ELISA检测,同时将其检测结果与免疫亲和层析高效液相色谱法（IAC-HPLC）进行比对,胶体金免疫层析试纸条与IAC-HPLC检测结果符合率为90％,间接竞争ELISA与IAC-HPLC检测结果相关系数达0．996,表明本研究所改进的样品前处理方法能提高免疫检测准确度,具有很好的应用性。     

荧光免疫定量法即时检测谷物中黄曲霉毒素B1

目的 基于时间分辨荧光免疫层析技术,研制快速定量检测试纸条,用于粮谷物中黄曲霉毒素B1的检测。方法 以荧光微球为标记物,采用DNP独立质控体系和竞争法检测原理,构建了粮谷物中黄曲霉毒素B1荧光免疫定量即时检测（point-of-care testing ,POCT）方法。评价其准确度、重复性、特异性、与仪器确证方法符合度。结果 该方法最适条件为：pH7.8硼酸盐（BB）活化,pH7.5磷酸盐（PB）偶联,微球抗体质量比为5:4,抗原抗体质量比为50:1。检测限在0.299 ng/g -0.997 ng/g之间,定量限在0.544 ng/g -2.663 ng/g。代表性样本准确度为92.2%-111%,重复性为3.2%-7.5%。除与黄曲霉毒素B2有交叉反应（6.1%）,与其他类似物无（<0.1%）。该法检测结果与仪器确证法一致性好,在-12.5%—15.9%之间。结论 提供了一种准确、快速、定量、灵敏、便捷、适合现场检测粮谷物中黄曲霉毒素B1的时间分辨荧光免疫检测试纸条。     

黄曲霉毒素B1免疫层析试纸条和ELISA试剂盒中花生粕样本适用性优化

本实验对比了商品化酶联免疫吸附法、荧光免疫层析快速定量法和胶体金快速免疫层析快速定量法中不同提取溶剂对花生粕中黄曲霉毒素B₁检测结果的影响,旨在商品化黄曲霉毒素B₁的快速检测产品中增加花生粕样本的适用性,以满足食品加工企业、饲料加工企业以及花生粕原料贸易商对花生粕中黄曲霉毒素B₁快速检测的需求。结果显示,商品化黄曲霉毒素B₁ ELISA检测检测试剂盒、黄曲霉毒素B₁荧光快速定量检测试纸条和黄曲霉毒素B₁胶体金快速定量检测试纸条在花生粕检测中的最优提取溶剂均为30%乙腈-水溶液,食品加工企业、饲料加工企业以及花生粕原料贸易可使用此方法对花生粕中黄曲霉毒素B₁进行快速检测。     

定性测定羊奶中黄曲霉毒素M1残留高灵敏度检测卡的研制

研制定性测定羊奶中黄曲霉毒素M1残留的高灵敏度检测卡.在硝酸纤维素膜上预包被黄曲霉毒素M1全抗原作为检测线,以及预包被羊抗鼠二抗作为质控线.将抗黄曲霉毒素M1单克隆抗体用胶体金标记作为金标垫.将样品垫、金标垫、预包被黄曲霉毒素M1的硝酸纤维素膜以及吸水垫依次粘贴在PVC板上,切割成试纸条并组装成检测卡.结果表明:该检测卡的检测限为0.5μg/L,对黄曲霉毒素M1的交叉反应率为100％,而与同族的其他物质的交叉反应率小于5％.假阳性率小于5％,假阴性率为0％,检测卡常温储存,保质期18个月.本研究的检测卡,可快速准确地应用于羊奶中黄曲霉毒素M1残留的定性检测,为快速准确地测定羊奶中黄曲霉毒素M1残留提供了依据.     

一种快速检测呕吐毒素的胶体金试纸条

利用胶体金免疫层析法,研制了一种能够快速检测黄豆中呕吐毒素含量的试纸条。通过试验,胶体金试纸条对呕吐毒素的检测限是1μg/g,对玉米赤霉烯酮毒素、黄曲霉毒素B1、赭曲毒素A等无交叉反应,假阳性率和假阴性率均为0,检测时间为10min。试纸条能够准确、可靠、简便、快速地检测黄豆中残留的呕吐毒素,适合大量样品的现场检测。     

Determination of aflatoxins in eggs,milk, meat and meat products using HPLC fluorescent and UV detectors

Raw and pasteurised sheep’s, cow’s and goat’s milk, eggs, and beef samples from different local markets in Jordan were collected during a period of 5 months (January through May 2007) and examined for aflatoxins B1(AFB1), B2(AFB2), G1(AFG1), G2(AFG2), M1(AFM1) and M2(AFM2). The samples were analysed with high performance liquid chromatography (HPLC) using UV and Fluorescent detectors. The analysed samples of milk collected in January were found to contain 0.56 μg L⁻¹ AFM1 and 0.1 μg L⁻¹ AFM2 whilst, the concentration of AFM1 and AFM2 was < 0.05 μg L⁻¹ for milk samples collected between March and May. The AFB1, AFB2, AFG1 and AFG2 contents in the analysed food products ranged from 1.10 to 8.32 μg L⁻¹ and 0.15 to 6.36 μg L⁻¹ in imported and fresh meat samples collected during March, respectively. The mean recovery for the HPLC method was 92% to 109% and the quantification levels were 50 ng L⁻¹ for AFM1 and AFM2. The AFM1 was found in 10% of the tested samples with concentrations between 0.08 and 1.1 μg kg⁻¹ and AFM2 was only found in 1.82% of the tested samples with a level of 0.1 μg kg⁻¹. The AFM1 levels in the examined foods were higher than the maximum level of AFM1 in liquid milk set by the European Community and Codex Alimentarius of 50 ng L⁻¹.     

Natural co-occurrence of aflatoxin B1, fumonisin B1 and ochratoxin A in barley and corn foods from Korea

A survey for aflatoxin B₁ (AFB₁), fumonisin B₁ and ochratoxin A (OTA) was conducted on 127 samples that included 30 food-grade barley, 32 barley foods, 18 food-grade corn and 47 corn foods, randomly collected during 1998-99 in Seoul, Korea. The presence of mycotoxins was analysed by direct competitive enzyme-linked immunosorbent assay (ELISA), and most of the positive samples from ELISA were confirmed using high-performance liquid chromatography (HPLC). Recoveries of AFB₁ and OTA spiked at 10 ng g -1 and FB₁ spiked at 50 ng g^-1 were 106, 87 and 105% by ELISA, whereas those by HPLC were 80, 79 and 84%, respectively. Detection limits by ELISA for AFB₁, FB₁ and OTA were 1, 5 and 5 ng g^-1, and those by HPLC were 0.6, 35 and 1 ng g^-1. Naturally occurring AFB₁, FB₁ and OTA were found in 4/32 (12%), 2/32(6%) and 4/32 (12%) samples of barley foods with an average of 26, 16 and 9 ng g^-1, respectively. AFB₁ and FB₁ in corn foods were detected in 4/47 (8%) and 9/47 (19%) samples with the average being 20 and 74 ng g^-1, while no OTA was found in any corn foods samples. No AFB₁, FB₁ or OTA was detected in any of food-grade barley and corn samples. This is the first report on the natural co-occurrence of AFB₁ and FB₁ in barley and corn foods as well as on surveillance of OTA in Korea.     

Determination of the variability of both hydrophilic and lipophilic toxins in endemic wild bivalves and carnivorous gastropods from the Southern part of Chile

The aim of this study was to analyse and determine the composition of paralytic shellfish poisoning (PSP) toxins and lipophilic toxins in the Region of Aysén, Chile, in wild endemic mussels (Mytilus chilensis, Venus antiqua, Aulacomya ater, Choromytilus chorus, Tagelus dombeii and Gari solida) and in two endemic carnivorous molluscs species (Concholepas concholepas and Argobuccinum ranelliforme). PSP-toxin contents were determined by using HPLC with fluorescence detection, while lipophilic toxins were determined by using LC-MS/MS. Mean concentrations for the total of PSP toxins were in the range 55–2505 μg saxitoxin-equivalent/100 g. The two most contaminated samples for PSP toxicity were bivalve Gari solida and carnivorous Argobuccinum ranelliforme with 2505 ± 101 and 1850 ± 137 μg saxitoxin-equivalent/100 g, respectively (p < 0.05). The lipophilic toxins identified were okadaic acid, dinophysistoxin-1 (DTX-1), azaspiracid-1 (AZA-1), pectenotoxin-2 (PTX-2) and yessotoxins (YTX). All analysed molluscs contained lipophilic toxins at levels ranging from 56 ± 4.8 to 156.1 ± 8.2 μg of okadaic acid-equivalent/kg shellfish together with YTX at levels ranging from 1.0 ± 0.1 to 18 ± 0.9 μg of YTX-equivalent/kg shellfish and AZA at levels ranging from 3.6 ± 0.2 to 31 ± 2.1 μg of AZA-equivalent/kg shellfish. Furthermore, different bivalves and gastropods differ in their capacity of retention of lipophilic toxins, as shown by the determination of their respective lipophilic toxins levels. In all the evaluated species, the presence of lipophilic toxins associated with biotransformation in molluscs and carnivorous gastropods was not identified, in contrast to the identification of PSP toxins, where the profiles identified in the different species are directly related to biotransformation processes. Thus, this study provides evidence that the concentration of toxins in the food intake of the evaluated species (Bivalvia and Gastropoda class) determines the degree of bioaccumulation and biotransformation they will thereafter exhibit.     

麦绿素对实验性高脂血症大鼠血脂及MDA、SOD、ET-1、NO的影响

研究麦绿素对食饵性高脂血症大鼠血脂及血浆丙二醛(MDA)、超氧化物歧化酶(SOD)、内皮素1(ET-1)、一氧化氮(NO)的影响。SD大鼠32只,随机均分为四组,即正常八周对照组(8NC)、高脂模型八周组(8HF)、高脂模型四周后再加低剂量麦绿素治疗四周组(BG-L)和高脂模型四周后再加高剂量麦绿素治疗四周组(BG-H)。检测大鼠血脂及血浆MDA、SOD、ET-1、NO的变化。结果表明,麦绿素治疗组同高脂模型八周组比较,血浆总胆固醇(TC)和低密度脂蛋白胆固醇(LDL-C)的含量降低,高密度脂蛋白胆固醇(HDL-C)的含量升高;血浆SOD活性和NO含量明显升高,而MDA和ET-1含量明显降低;提示麦绿素具有降血脂、抗氧化和改善血管内皮功能的作用。     

Effect of calcium and calmodulin antagonists on ethylene biosynthesis in tomato fruits

The aim of this work was to elucidate the mode of action of calcium in calcium-inhibited ethylene biosynthesis. In one experiment, mature green tomatoes were treated with 1·5% (w/v) CaCl₂+60 μM N-6(aminohexyl)-1-naphthalenesulphonamide (W5). 1·5% (w/v) CaCl₂+60 μM N-6(aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7), 1·5% (w/v) CaCl₂ alone or distilled water (control). The control and the W7 treatment had much higher ethylene production than either the CaCl₂ treatment alone or the CaCl₂+W5 treatment. In another experiment, similar tomatoes were treated with 1·5% (w/v) CaCl₂+15 μM trifluoperazine (TFP). 1·5% (w/v) CaCl₂ only, or distilled water (control). The treatment with 1·5% CaCl₂+15 μM TFP gave higher ethylene production than 1·5% (w/v) CaCl₂ without TFP, and nearly the same level as the control towards the end of the experimental period. For enzyme and intermediate metabolite assays, mature green tomatoes were treated with 1·5% (w/v) CaCl₂ plus either W5 or W7. W5 treatment had a lower ethylene forming enzyme (EFE) activity, higher 1-aminocyclopropane-1-carboxylic acid (ACC) concentration, similar levels of ACC synthase activity and 1-(malonylamino)-cyclopropane-1-carboxylic acid concentration, compared to the control and W7 treatment. As W7 is a more potent calmodulin antagonist than W5, these results suggest a calcium-calmodulin inactivation of EFE activity. © 1998 SCI.     

基于尿苷二磷酸葡萄糖醛酸转移酶1解毒通路探讨枸杞汁对邻苯二甲酸二(2-乙基己基)酯代谢的干预作用

目的:通过大鼠毒性干预实验观察枸杞汁对邻苯二甲酸二(2-乙基己基)酯(di-(2-ethylhexyl)phthalate,DEHP)染毒大鼠肝脏中尿苷二磷酸葡萄糖醛酸转移酶1(uridine diphosphate glucuronosyltransferase 1,UGT1)活性及代谢解毒的影响,探讨枸杞汁对DEHP代谢的干预效果。方法:60只雄性大鼠随机分为对照组、DEHP组和枸杞汁干预组,每组20只,DEHP组和干预组在DEHP一次染毒(3 000 mg/kg mb)后连续7 d分别灌胃生理盐水和枸杞汁,对照组则给予同等体积的芝麻油或生理盐水,期间每天收集大鼠24 h尿液。在染毒1、3、5、7 d后各组分别随机处死5只。采用试剂盒检测肝脏UGT1的活力,高效液相色谱法检测血清中的邻苯二甲酸单(2-乙基己基)酯(mono-(2-ethylhexyl)phthalate,MEHP)以及尿液MEHP和邻苯二甲酸(phthalic acid,PA)含量。结果:枸杞汁干预组大鼠第5天UGT1活性显著高于DEHP组和对照组(P0.05),且相对于DEHP组血清中MEHP含量减少而尿液中MEHP和PA含量增加。结论:枸杞汁可能通过提高UGT1活力促进了DEHP的代谢与排泄,发挥了解毒效应,将来可能作为一种预防或对抗邻苯二甲酸酯类物质或其他有毒化学物潜在危害的保健药材或功能食品用于人群的健康防护。     

Saxitoxins and okadaic acid group: accumulation and distribution in invertebrate marine vectors from Southern Chile

Harmful algae blooms (HABs) are the main source of marine toxins in the aquatic environment surrounding the austral fjords in Chile. Huichas Island (Aysén) has an history of HABs spanning more than 30 years, but there is limited investigation of the bioaccumulation of marine toxins in the bivalves and gastropods from the Region of Aysén. In this study, bivalves (Mytilus chilenses, Choromytilus chorus, Aulacomya ater, Gari solida, Tagelus dombeii and Venus antiqua) and carnivorous gastropods (Argobuccinum ranelliformes and Concholepas concholepas) were collected from 28 sites. Researchers analysed the accumulation of STX-group toxins using a LC with a derivatisation post column (LC-PCOX), while lipophilic toxins (OA-group, azapiracids, pectenotoxins and yessotoxins) were analysed using LC-MS/MS with electrospray ionisation (+/–) in visceral (hepatopancreas) and non-visceral tissues (mantle, adductor muscle, gills and foot). Levels of STX-group and OA-group toxins varied among individuals from the same site. Among all tissue samples, the highest concentrations of STX-group toxins were noted in the hepatopancreas in V. antiqua (95 ± 0.1 μg STX-eq 100 g^?1), T. dombeii (148 ± 1.4 μg STX-eq 100 g^?1) and G. solida (3232 ± 5.2 μg STX-eq 100 g^?1; p < 0.05); in the adductor muscle in M. chilensis (2495 ± 6.4 μg STX-eq 100 g^?1; p < 0.05) and in the foot in C. concholepas (81 ± 0.7 μg STX-eq 100 g^?1) and T. dombeii (114 ± 1.2 μg STX-eq 100 g^?1). The highest variability of toxins was detected in G. solida, where high levels of carbamate derivatives were identified (GTXs, neoSTX and STX). In addition to the detected hydrophilic toxins, OA-group toxins were detected (OA and DTX-1) with an average ratio of ≈1:1. The highest levels of OA-group toxins were in the foot of C. concholepas, with levels of 400.3 ± 3.6 μg OA eq kg^?1 (p < 0.05) and with a toxic profile composed of 90% OA. A wide range of OA-group toxins was detected in M. chilensis with a toxicity < 80 μg OA eq kg^?1, but with 74% of those toxins detected in the adductor muscle. In all evaluated species, there was no detection of lipophilic toxins associated with biotransformation in molluscs and carnivorous gastropods. In addition, the STX-group and OA-group toxin concentrations in shellfish was not associated with the presence of HAB. The ranking of toxin concentration in the tissues of most species was: digestive glands > mantle > adductor muscle for the STX-group toxins and foot > digestive gland for the OA-group toxins. These results gave a better understanding of the variability and compartmentalisation of STX-group and OA-group toxins in different bivalve and gastropod species from the south of Chile, and the analyses determined that tissues could play an important role in the biotransformation of STX-group toxins and the retention of OA-group toxins.     

鲜食葡萄无公害贮藏保鲜处理

采用乙烯受体抑制剂1-甲基环丙烯(1-MCP)不同浓度、臭氧(O3)不同浓度和处理时间相结合处理我国南方产区的巨峰葡萄,并对其冷藏期保鲜效果进行研究.结果表明:在处理环境温度为10℃,相对湿度(RH)为60%～80%条件下,臭氧处理浓度和处理时间交互作用影响腐烂率,两者与腐烂率呈显著负相关(r=-0.9908),其中高浓度、长时间臭氧(O3)处理对供试鲜食葡萄的防腐保鲜效果更好一些,巨峰葡萄落粒率与乙烯受体抑制剂1-MCP浓度相关性不大(r=-0.042 8),可见该供试葡萄衰老引起的落粒可能不受乙烯直接影响,或果梗衰老引起的离层落粒可能不是该鲜食葡萄落粒的主要诱因.     

1-MCP处理对杏鲍菇采后生理和结构变化的影响

以杏鲍菇为试材,分别研究1-甲基环丙烯（1-methylcyclopropene,1-MCP）的不同剂量及不同处理时间对杏鲍菇采后质量损失率、多酚氧化酶（PPO）活性及菇体断面微细结构的影响。结果表明：当1-MCP的剂量为0.3 μL/L时,质量损失率和PPO活性在贮藏末期分别较对照组下降了5.94%和37.38%,菇体断面微细结构较贮藏初期变化最小;当1-MCP的处理时间为24 h时,质量损失率和PPO活性在贮藏末期分别较对照组下降了5.42%和33.98%,贮藏前后菇体断面微细结构变化最小。实验证明了1-MCP的最适处理剂量为0.3 μL/L、最适处理时间为24 h。最适条件下的1-MCP处理能有效地减缓果实质量损失率和褐变程度,减小菇体断面微细结构变化,有利于延长杏鲍菇的贮藏期,具有明显的保鲜效果。