赭曲霉毒素A单克隆抗体免疫亲和柱的制备

制备了赭曲霉毒素A(OTA)单克隆抗体免疫亲和柱,并用间接竞争ELISA和HPLC法评价了免疫亲和柱的性能,其柱容量(结合OTA的能力)约为200ng,加标回收率为90.38%～100.1%,可反复使用3次。 建立了免疫亲和柱-HPLC联用分析谷物中OTA的方法,最低检出限为0.2μg/kg,线性范围为0.6～400μg/kg,OTA加标量为1～10μg/kg时谷物样品中的回收率为78.7%～87.1%,变异系数小于6.5%。用此法检测了大米、小麦、玉米和玉米饲料等15份市售样品,检出率为46.7%,其中OTA的最高含量为0.785μg/kg。     

木薯叶粉中赭曲霉毒素A快速检测方法的建立

目的 开发快速检测方法检测木薯叶粉中的赭曲霉毒素A(Ochratoxins A, OTA)。方法 利用单克隆抗体和酶标抗原, 建立了竞争酶联免疫吸附测定法(enzyme-linked immunosorbent assay, ELISA)快速检测方法。用不同浓度的氯化钠甲醇水溶液、乙腈和磷酸盐缓冲液等作为样本提取液提取木薯叶粉中OTA, 用活性炭和稀释法减少样本基质干扰。结果 ELISA灵敏度为0.2 ng/mL, IC50为0.878 ng/mL。木薯叶粉用含氯化钠的70%甲醇水溶液提取, 离心后取上清5倍稀释, 样本回收率80%~121%, 检测限为5 μg/kg, 满足国标规定食品中OTA不超过5 μg/kg的要求, 检测时间为30 min, 一次可批量检测90个样本。结论 用本研究建立的ELISA方法检测木薯叶粉及其食品中的OTA含量结果准确、操作简单、成本低、时间短、重复性好, 适合基层批量筛查。     

高效液相法同时检测谷物中玉米赤霉烯酮和赭曲霉毒素A

该研究开发一种快速、灵敏同时检测玉米、小麦和稻米中玉米赤霉烯酮(zearalenone,ZEN)和赭曲霉毒素A(ochratoxin A,OTA)的高效液相色谱检测方法。将样品采用乙腈/水(80/20,体积比),200 r/min,30℃振荡提取30 min后,经Oasis PRiME HLB固相萃取柱净化后,上样检测。该方法ZEN和OTA检测限(limit of detection,LOD)为3.7μg/kg和0.11μg/kg,定量限(quantification Limit,LOQ)为12.25μg/kg和0.38μg/kg,线性范围分别为10μg/kg^2000μg/kg和0.2μg/kg^200μg/kg,加标样品中不同浓度的ZEN和OTA回收率为83.0%~101.3%,日内精密度和日间精密度分别为3.12%~7.03%和3.57%~9.3%。该方法适用于玉米、小麦和稻米中ZEN和OTA的同时检测。     

检测赭曲霉毒素A(OTA)的酶联免疫吸附法(ELISA)体系的建立

本文分别以自制抗-OTA兔血清抗体(IgG)及鸡卵黄抗体(IgY)就影响检测OTA的ELISA(酶联免疫吸附法)体系因素进行了探讨,建立了检测OTA的ELISA方法。鸡卵黄抗体的最佳ELISA操作参数为:包被抗原浓度为7.5μg/ml,封阻剂浓度为2.5%,抗体稀释倍数为1000,酶标二抗稀释倍数为14000;兔血清抗体的最佳ELISA操作参数为:包被抗原浓度为2.5μg/ml,封阻剂浓度为2.5%,抗体稀释倍数为8000,酶标二抗稀释倍数为10000。IgG和IgY的检测灵敏度分别为10ng/ml和1ng/ml。     

谷物中赭曲霉毒素A化学发光酶免疫分析法的建立

建立了间接竞争化学发光酶免疫法检测赭曲霉毒素A(ochratoxin A,OTA),该方法IC50为112 pg/mL,检出限是2.47 pg/mL,平均批内和批间变异系数分别为7.03%和14.7%。在大米和小麦样本中添加浓度1.5～6μg/kg的OTA标品,平均回收率在66.97%～97.96%之间,与其他常见真菌毒素未见交叉反应。将该方法应用于30份谷物样本(包括20份大米样本和10份小麦样本)中OTA的检测,检测结果与商品化ELISA试剂盒的相关系数R2=0.942 4。该方法简单、灵敏、快速、准确适用于谷物中OTA的检测。     

高效液相色谱法测定粮食及其制品中赭曲霉毒素A提取条件的优化

目的 优化高效液相色谱法测定粮食及其制品中赭曲霉毒素A(ochratoxin A, OTA)的提取条件。 方法 样品通过乙腈-水(60:40, V:V)提取, 高速均质2 min或振荡提取20 min, 采用赭曲霉毒素A免疫亲和柱浓缩净化、高效液相色谱-荧光检测器定量检测。结果 选用OTA自然污染的粮食样品优化方法更符合实际检测。60%乙腈-水的提取效率优于80%的甲醇-水, 且振荡提取20 min, 一次提取即可提取样品中99%以上的OTA。4种粮食及其制品中OTA在0.2~50 ?g/kg范围内线性关系良好, 相关系数为r2=0.9997。加标回收率为80.7%~108.0%, 变异系数为1.6%~7.5%, 检出限为0.06 μg/kg, 定量限为0.2 μg/kg。采用此方法参加比利时国际能力验证, |Z|<2, 结果满意。同时, 基于本方法组织的国际实验室环形验证中, 粮食及其制品中OTA测定值的统计结果符合《食品法典委员会程序手册》(第二十版)中关于联合验证中HorRat在2之内的规定。 结论 优化后的方法灵敏度高、准确性好, 适合粮食及其制品中赭曲霉毒素A的精准测定。     

检测葡萄酒中赭曲霉毒素A的SPE-HPLC方法优化

从固相萃取的上样体积、淋洗液体积以及高效液相色谱的洗脱程序等方面,优化并建立了检测葡萄酒中赭曲霉毒素A的(SPEHPLC)方法.采用10mL酒样与10mL水等体积混合上样,使用2mL水淋洗后再用2mL甲醇/水(60∶40,v/v)溶液的最佳淋洗条件,HPLC 检测首次采用梯度洗脱,优化后方法的加标回收率为94.6%～99.5%,相对标准偏差为0.36%～3.01%.6个市售葡萄酒样品检测表明,OTA阳性率为66.7%,平均含量为0.46μg/L,证明本方法能够排除杂质峰干扰,提高准确度,可以满足葡萄酒中赭曲霉毒素(OTA)的定量检测要求.     

河南省市售小麦粉微生物及真菌毒素污染状况研究

为了解河南省市售小麦粉的安全状况,收集整理河南省市售小麦粉。采用Filmplate~(TM)测试片法测定样品的菌落总数、霉菌和酵母菌计数及大肠菌群,采用酶联免疫法(Enzyme–Linked Immuno Sorbent Assay,ELISA)测定样品中呕吐毒素(DON)、黄曲霉毒素B_1(AFB_1)及赭曲霉毒素A(OTA)三种常见真菌毒素的含量。结果显示:小麦粉样品受微生物污染面积较广,但程度可控。菌落总数、霉菌酵母菌数、大肠菌群数主要集中范围分别是10~2~10~4、10~2~10~3、10~2~10~3 CFU/g;DON、AFB_1及OTA含量超限率分别为0.00%、13.85%、7.69%,检出率依次为69.23%、52.31%及84.62%,污染程度不同,污染范围均较为广泛。     

食品中硼酸(硼砂)快速测定方法研究

采用乙腈作为萃取溶剂,姜黄素分光光度法测定,研究以微波法提取食品中的硼酸(硼砂)。结果表明:2.5%的硫酸溶液提取效果理想,当样品溶液中加入氯化钠并使之饱和,乙腈能够达到理想的分离效果,且方法提取时间由传统方法的10 min缩短为1 min,加标回收率为97.3%～103.5%,相对标准偏差(RSD)小于5%。     

在线免疫亲和固相萃取-高效液相色谱法快速测定食品中赭曲霉毒素A

目的建立在线免疫亲和固相萃取-高效液相色谱快速测定食品中赭曲霉毒素A(ochratoxin A,OTA)的分析方法。方法谷物、豆类样品采用乙腈-水(80:20,V:V)提取,葡萄酒类样品采用NaHCO_3/NaCl/水溶液(0.4:15:100,m:m:V)提取,3%Tween-20(m:V)溶液稀释后,直接经RIDA~?CREST ICE在线免疫亲和固相萃取系统净化-高效液相色谱荧光检测OTA含量。以乙酸铵-乙腈为流动相洗脱,流速1.0 mL/min,C_(18)色谱柱(150 mm×4.6mm,5μm)分离测定。结果方法检出限分别为0.020μg/kg(豆类、谷物)和0.050μg/kg(葡萄酒类);在OTA的浓度为12.5~500 ng/L的范围内线性关系良好,相关系数r~2为0.9995;日内加标回收率为87.4%~109.8%,相对标准偏差0.50%~4.80%。结论该方法分析速度快,灵敏度高,重现性好,可满足快速准确测定大批量食品样品中OTA含量的需要。     

Determination of Ochratoxin A in Bread: Evaluation of Microwave-Assisted Extraction Using an Orthogonal Composite Design Coupled with Response Surface Methodology

An analytical method using microwave-assisted extraction (MAE) and liquid chromatography (LC) with fluorescence detection (FD) for the determination of ochratoxin A (OTA) in bread samples is described. A 2⁴ orthogonal composite design coupled with response surface methodology was used to study the influence of MAE parameters (extraction time, temperature, solvent volume, and stirring speed) in order to maximize OTA recovery. The optimized MAE conditions were the following: 25 mL of acetonitrile, 10 min of extraction, at 80 °C, and maximum stirring speed. Validation of the overall methodology was performed by spiking assays at five levels (0.1–3.00 ng/g). The quantification limit was 0.005 ng/g. The established method was then applied to 64 bread samples (wheat, maize, and wheat/maize bread) collected in Oporto region (Northern Portugal). OTA was detected in 84 % of the samples with a maximum value of 2.87 ng/g below the European maximum limit established for OTA in cereal products of 3 ng/g.     

Fluorescence Polarization Immunoassay for Rapid, Accurate and Sensitive Determination of Ochratoxin A in Wheat

A sensitive and accurate fluorescence polarization (FP) immunoassay has been developed for the determination of ochratoxin A (OTA) in naturally contaminated wheat samples. A fluorescein-labeled OTA tracer was synthesized, and its binding response with three monoclonal antibodies was tested. The most sensitive competitive FP immunoassay showed an IC₅₀ value of 0.48 ng/mL with a negligible cross-reactivity for ochratoxin B (1.7 %) and no cross-reactivity with other mycotoxins commonly occurring in wheat. The wheat sample was extracted with acetonitrile/water (60:40, v/v) and purified by a rapid solid-phase extraction procedure using an aminopropyl column prior to the FP immunoassay. The overall time of analysis was less than 20 min. The average recovery from spiked wheat samples (3 to 10 μg/kg) was 87 %, with relative standard deviations generally lower than 6 %. Limits of detection and quantification were 0.8 and 2.0 μg/kg, respectively. The trueness of the method was assessed by using two reference materials for OTA showing good accuracy and precision. A good correlation (r?=?0.995) was observed between OTA contamination of 19 naturally contaminated wheat samples analyzed by both FP immunoassay and high-performance liquid chromatography/immunoaffinity clean-up used as reference method. These results show that the developed FP method is suitable for high-throughput screening, as well as for reliable quantitative determination of OTA in wheat at level far below the EU regulatory limits.     

Development and application of analytical methods for the determination of mycotoxins in organic and conventional wheat.

The aim of this study was to develop a multicomponent analytical method for the determination of deoxynivalenol (DON), ochratoxin A (OTA) and zearalenone (ZEN), nivalenol (NIV), 3-acetyl-DON (3-acDON), 15-acetyl-DON (15-acDON), zearalenol (ZOL) and citrinin (CIT) in wheat. It also aimed to survey the presence and amounts of DON, OTA and ZEN in Belgian conventionally and organically produced wheat grain and in wholemeal wheat flours. After solvent extraction, an anion-exchange column (SAX) was used to fix the acidic mycotoxins (OTA, CIT), whilst the neutral mycotoxins flowing through the SAX column were further purified by filtration on a MycoSep cartridge. OTA and CIT were then analysed by high-performance liquid chromatography (HPLC) using an isocratic flow and fluorescence detection, while the neutral mycotoxins were separated by a linear gradient and detected by double-mode (ultraviolet light fluorescence) detection. The average DON, ZEN and OTA recovery rates from spiked blank wheat flour were 92, 83 and 73% (RSDR = 12, 10 and 9%), respectively. Moreover, this method offered the respective detection limits of 50, 1.5 and 0.05 microg kg-1 and good agreement with reference methods and inter-laboratory comparison exercises. Organic and conventional wheat samples harvested in 2002 and 2003 in Belgium were analysed for DON, OTA and ZEN, while wholemeal wheat flour samples were taken from Belgian retail shops and analysed for OTA and DON. Conventional wheat tended to be more frequently contaminated with DON and ZEN than organic samples, the difference being more significant for ZEN in samples harvested in 2002. The mean OTA, DON and ZEA concentrations were 0.067, 675 and 75 microg kg-1 in conventional samples against 0.063, 285 and 19 microg kg-1 in organically produced wheat in 2002, respectively. Wheat samples collected in 2003 were less affected by DON and ZEN than the 2002 harvest. Organic wholemeal wheat flours were more frequently contaminated by OTA than conventional samples (p < 0.10). The opposite pattern was shown for DON, organic samples being more frequently contaminated than conventional flours (p < 0.10).     

Determination of ochratoxin A in wheat after clean-up through a DNA aptamer-based solid phase extraction column

A DNA aptamer with high affinity and specificity to ochratoxin A (OTA) was conjugated to a coupling gel and used as sorbent for the preparation of solid phase extraction (SPE) columns. The SPE columns packed with 300 μl oligosorbent (24 nmol DNA) showed a linear (r = 0.999) behaviour in the range of 0.4–500 ng OTA. After optimisation of the extraction step, SPE columns were used for clean-up of OTA from wheat prior to liquid chromatographic (HPLC) analysis with fluorescence detection (FLD). Average recoveries from wheat samples spiked at levels of 0.5–50 ng/g ranged from 74% to 88% (relative standard deviation <6%) with limits of detection and of quantification of 23 and 77 pg/g, respectively. The comparative HPLC/FLD analyses of 33 naturally contaminated durum wheat samples cleaned-up on both aptamer-SPE and immunoaffinity (IMA) columns showed a good correlation (r = 0.990). Aptamer-SPE columns could be re-used up to five times without any loss of performance.     

Ochratoxin A in sultanas from Turkey I: Survey of unprocessed sultanas from vineyards and packing-houses.

A method for the determination of ochratoxin A (OTA) in sultanas from Turkey using extraction with a sodium bicarbonate solution (2% NaHCO3) followed by immunoaffinity clean-up and liquid chromatography with fluorescence detection was used to assess the frequency of occurrence and level of OTA. In-house validation was carried out with spiked samples at levels of 0.15, 1.5, 5.0 and 10 microg kg-1 and average recoveries were 91, 93, 87 and 89%, respectively. The limits of detection and limit of quantification in Turkish sultanas were 0.026 and 0.09 microg kg-1, respectively. A survey for the presence of OTA was carried out on 264 unprocessed sultana samples during the production seasons between 1998 and 2000 collected annually from vineyards and from packing-houses. The analyses of unprocessed sultanas showed that 32.2% of the total number of samples contained no detectable OTA, whereas 9.8% of sultana samples had OTA concentrations above 10 microg kg-1, and the remaining 58% had levels within the range 0.026-10 microg kg-1. There were big differences in median concentrations between years. Considering the year of production, it appears that sultanas produced in 1998 and 2000 showed the lowest incidence of OTA contamination (median<0.02 microg kg-1), whereas 2002 showed the highest incidence (median=4.3 microg kg-1). The overall mean OTA concentration was calculated as 3.4 microg kg-1, and the overall median as 0.9 microg kg-1. Among the samples analysed, the highest detected level of OTA was 54 microg kg-1.     

Two-dimensional thin-layer chromatographic method for the analysis of ochratoxin A in green coffee

A low-cost thin-layer chromatographic method has been developed for the presumptive measurement of ochratoxin A (OTA) at 5 microg/kg in green coffee beans. The analytical method consisted of extracting OTA by shaking the beans with a mixture of methanol and aqueous sodium bicarbonate solution, which was then purified by liquid-liquid partition into toluene. OTA was separated by normal-phase two-dimensional thin-layer chromatography and detected by visual estimation of fluorescence intensity under a UV lamp at 365 nm. The chromatography solvents were toluene-methanol-formic acid (8:2:0.03) for the first development and petroleum ether-ethyl acetate-formic acid (8:10:1) for the second dimension development. This method was tested with uncontaminated green coffee bean samples spiked with an OTA standard at four different concentrations (5, 10, 20, and 30 microg/kg). The method is rapid, simple, and very easy to implement in coffee-producing countries. It is highly selective and does not involve the use of chlorinated solvents in the sample extraction step. This inexpensive method has been applied to different types of green coffee samples from various countries (Zimbabwe, Brazil, India, Uganda, Colombia, and Indonesia) and different manufacturers, and no OTA below the detection limit of 5 microg/kg was detected in any samples analyzed.     

真菌毒素在赤霉病小麦研磨及其湿法加工中的分布研究

小麦赤霉病不仅会导致粮食减产,更会引起多种真菌毒素的高污染风险。将染病小麦进行实验室制粉并湿法分离统粉中的粗淀粉、谷朊粉,采用酶联免疫吸附测定法(Enzyme-Linked Immuno Sorbent Assay,ELISA)测定各系统的呕吐毒素(Deoxynivalenol,DON)、黄曲霉毒素B1(Aflatoxin B1,AFB1)、玉米赤霉烯酮(Zearalenone,ZEN)以及赭曲霉毒素A(Ochratoxin A,OTA)的含量,以探究赤霉病小麦在制粉及其湿法加工中四种常见真菌毒素的分布变化规律。结果显示:研磨制粉及湿法加工对真菌毒素的分布影响显著。制粉加工后,皮磨和心磨系统粉的DON、AFB1、ZEN和OTA含量比原粮显著降低,降低率分别为1.38~16.24%、20.47~71.77%及26.71~69.51%;湿法加工产物中,DON消减为相应统粉的4.88~12.11%,AFB1与OTA浓缩富集,富集率分别可达统粉的2.55、3.65倍,粗淀粉中ZEN消减为统粉的12.70~15.83%,谷朊粉则富集为统粉的4.11倍。研究表明,在工业生产中,可根据赤霉病小麦的感染类型及程度,适当选用研磨或湿法加工等合适的方法加工处理。     

Influence of roasting and different brewing processes on the ochratoxin A content in coffee determined by high-performance liquid chromatography-fluorescence detection (HPLC-FLD)

A rapid and reliable procedure has been developed for the determination of ochratoxin A (OTA) in green and roasted coffee. The method consists of extraction of the sample with methanol-5% aqueous sodium hydrogen carbonate/1% PEG8000 (20:80), followed by immunoaffinity column (IAC) clean-up and, finally, high-performance liquid chromatography (HPLC) determination with fluorimetric detection. Mean recoveries for green and roasted coffee spiked at different levels ranging from 94 and 105% were obtained. The limit of determination (S/N = 3) was 0.032 ng g(-1) and the precision (within-laboratory relative standard deviation) was 6%. The method described has been used to assess the influence of roasting and different brewing processes on OTA content in commercial lots of green and roasted coffee. The results provided evidence that roasting led to a significant drop on OTA levels (65-100%). Also, the way coffee is prepared affects the OTA content: brewing using a Moka Express (Italian coffee) led to a significant reduction of OTA concentration (50-75%) since hot water stays in contact with coffee for a short time. On the contrary, Turkish coffee-making (infusion for about 10 min) cause poor reduction in OTA.