脱氧雪腐镰刀菌烯醇酶联免疫检测方法的建立

应用抗脱氧雪腐镰刀菌烯醇单克隆抗体12D1建立了竞争间接ELISA方法,用于检测小麦、玉米中的脱氧雪腐镰刀菌烯醇(DON),最低检出限为20ng/mL,检测范围为20￣460ng/mL。用蒸馏水提取掺合DON(250￣4000ng/g)的小麦样品,回收率为82.1%￣96.6%,变异系数为0.6%￣7.1%。     

脱氧雪腐镰刀菌烯醇时间分辨荧光免疫检测体系适用性评价

目的研究已建立的脱氧雪腐镰刀菌烯醇时间分辨荧先兊疫定量检测体系(包括脱氧雪腐镰刀菌烯醇时间分辨荧先兊疫层析检测卡和荧先定量检测仪)的适用性。方法检测20个阴性样本中脱氧雪腐镰刀菌烯醇含量,确定该检测体系的检出限和定量限;对6组含不同浓度脱氧雪腐镰刀菌烯醇的小麦阳性样本和6组含不同浓度脱氧雪腐镰刀菌烯醇的玉米阳性样本进行检测,确定方法的准确性和台间差;重复6次检测含中等浓度脱氧雪腐镰刀菌烯醇的阳性样本和连续12 h检测国家检测限浓度的阳性样本,确定系统的重复性和稳定性。结果该体系的检出限为154μg/kg,定量限为414μg/kg,幵且检测结果与液质联用方法定值结果无显著性差异,仪器台间差无显著差异;测值的精密度未超过国家标准规定要求。结论本研究验证的时间分辩荧先定量检测体系能准确检测玉米和小麦样本中脱氧雪腐镰刀菌烯醇含量。     

免疫亲和层析高效液相色谱法对小麦粉中脱氧雪腐镰刀菌烯醇的测定

目的：建立测定小麦粉中脱氧雪腐镰刀菌烯醇含量的免疫亲和层析高效液相色谱法。方法：小麦粉经粉碎处理,用水提取,通过免疫亲和柱上样、净化和洗脱后,用高效液相色谱仪测定小麦粉中脱氧雪腐镰刀菌烯醇的含量。结果：在100～5 000 ng·mL^-1,方法的线性关系良好,相关系数(R²)为0.999 8,检出限为48 ng·mL^-1,回收率为90.1%～109.6%,相对标准偏差为1.49%～2.20%。结论：该方法操作步骤简便,特异性强,精密度高,可以作为大批量分析小麦粉中脱氧雪腐镰刀菌烯醇含量的处理方案。     

粮食中脱氧雪腐镰刀菌烯醇风险预警研究进展

脱氧雪腐镰刀菌烯醇是小麦和玉米等粮食中常见的真菌毒素,受污染的粮食会严重影响人和动物健康,控制粮食产前镰刀菌感染和脱氧雪腐镰刀菌烯醇的积累是保障粮油食品安全的重要环节。当前,世界各国都十分重视粮食真菌毒素污染风险预警工作,粮食产前脱氧雪腐镰刀菌烯醇的污染预警研究近年来发展迅速,在脱氧雪腐镰刀菌烯醇积累的田间影响因素研究和建立产前风险预警模型中,都取得了一定进展。本文综述了脱氧雪腐镰刀菌烯醇风险预警的最新研究成果,旨在总结和整合现有的研究方法和成果,为开展我国粮食真菌毒素早期预警提供参考。     

高效液相色谱法测定粮谷类中脱氧雪腐镰刀菌烯醇

利用纯水溶解提取粮谷类样品中的脱氧雪腐镰刀菌烯醇,经过免疫亲和柱净化,并优化提取净化条件,通过高效液相色谱检测其含量。结果表明,采用2 mL滤液上样,经氮吹仪在40 ℃吹干后,用1 mL乙腈-水（20∶80,V/V）定容,在波长220 nm条件下进行检测,色谱峰分离效果良好。脱氧雪腐镰刀菌烯醇含量在20～500 ng/mL范围内线性关系良好（R2=0.999 8）,检出限2 μg/kg,精密度试验结果相对标准偏差（RSD）值为2.4%,回收率为87.6%～98.9%,RSD值为2.04%～2.68%,表明该方法具有良好的精密度和准确性,适用于粮谷类样品中脱氧雪腐镰刀菌烯醇含量的定量分析检测。     

脱氧雪腐镰刀菌烯醇免疫亲和层析柱的研制

利用本实验室研制的抗脱氧雪腐镰刀菌烯醇(DON)单克隆抗体研制了DON单克隆抗体免疫亲和柱,柱容量为1μg。小麦样品添加DON标品(1～3μg/g),用蒸馏水提取样品中DON,经免疫亲和柱净化、甲醇洗脱,HPLC检测,回收率大于88%,变异系数4.6%～7.4%。     

脱氧雪腐镰刀菌烯醇胶体金检测试纸的研制及应用

采用合成脱氧雪腐镰刀菌烯醇(Dexynivalenol,DON)人工抗原,制备抗脱氧雪腐镰刀菌烯醇单克隆抗体(mAb),并以此为基础建立胶体金免疫层析检测试纸,于胶体金读数仪配合使用,用于进行玉米和小麦样品中DON的快速定量检测。通过检测玉米和小麦样品,脱氧雪腐镰刀菌烯醇胶体金试纸方法检测限为87μg/kg,定量限为290μg/kg,线性范围400~5 000μg/kg,相关系数r=0.998。以国家标准(GB/T23503—2009)免疫亲和柱-高效液相色谱法检测样品的结果为认定值,胶体金检测法的准确度在80.7%~143.6%,变异系数范围4.7%~16.4%。采用配对t-检验分析,胶体金检测法与国标液相方法无显著性差异。     

免疫亲和柱净化—高效液相色谱法同时检测小麦中脱氧雪腐镰刀菌烯醇及其衍生物

建立脱氧雪腐镰刀菌烯醇(DON)及其衍生物3-乙酰基—脱氧雪腐镰刀菌烯醇(3-ADON)和15-乙酰基—脱氧雪腐镰刀菌烯醇(15-A-DON)的免疫亲和柱净化—高效液相色谱同时检测方法。小麦样品经超纯水提取、免疫亲和柱净化、吹干、定容后,用配有紫外检测器的高效液相色谱仪进行DON、3-A-DON和15-A-DON三种毒素的同时检测,并与液相色谱质谱法比对。结果表明:该方法检出限,DON、3-A-DON和15-A-DON均为100μg/kg,样品加标回收率范围86.6%~96.5%,变异系数小于10%,具有较好的准确性和精密度。该方法简单、快速、灵敏度高、选择性好,适用于小麦中DON、3-A-DON和15-A-DON的同时检测。     

流动注射化学发光法在线检测饮用水中脱氧雪腐镰刀菌烯醇

为应对突发性脱氧雪腐镰刀菌烯醇污染饮用水事件的发生,基于脱氧雪腐镰刀菌烯醇对鲁米诺—过氧化氢体系的促进作用,建立流动注射化学发光在线检测脱氧雪腐镰刀菌烯醇的新方法。在优化的试验条件下,该方法测定脱氧雪腐镰刀菌烯醇的线性范围为0.001~4.000 mg/L;检出限(S/N=3)为0.001 mg/L;相对标准偏差(RSD,n=11)为1.89%;不同加标水平下的加标回收率为67.33%~94.50%;相对标准偏差(n=3)小于2.34%。该方法具有灵敏度高、分析速度快及操作简便等优点,可应用于突发性脱氧雪腐镰刀菌烯醇污染的在线快速检测和应急预警。     

液质联用同时检测小麦中三种镰刀菌毒素

研究了小麦面粉中玉米赤霉烯酮、脱氧雪腐镰刀菌烯醇、雪腐镰刀菌烯醇三种镰刀菌毒素液相色谱-质谱联用同时检测的方法。小麦样品中的毒素提取液经Mycosep 226多功能净化柱净化后，以反相C18柱为色谱柱，甲醇、0．1％乙酸铵水溶液梯度洗脱为流动相，大气压化学电离离子源电离后质谱检测。该方法玉米赤霉烯酮、脱氧雪腐镰刀菌烯醇和雪腐镰刀菌烯醇标准溶液的提取离子峰面积与标品浓度分别在3～4500ng／mL、8～5000ng／mL范围内呈良好的线性关系，加标实验中三种毒素平均回收率在70．9％～93．7％之间，检出限分别为5、10、12ng／g，RSD均小于10％，具有很好的精密度。     

Interlaboratory study of LC-UV and LC-MS methods for the simultaneous determination of deoxynivalenol and nivalenol in wheat

To evaluate LC methods with UV or MS detection for simultaneous analysis of deoxynivalenol (DON) and nivalenol (NIV) in wheat, an interlaboratory study was conducted in 11 laboratories. DON and NIV were purified using a multifunctional column, and their concentrations were determined using LC-UV or LC-MS(/MS). No internal standards were used. Three fortified wheat samples (0.1, 0.5 and 1 mg/kg), one naturally contaminated wheat sample, and one blank wheat sample were used. The recoveries ranged from 90% to 110% for DON and from 76% to 83% for NIV. For DON, the relative standard deviations for repeatability (RSDr) ranged from 1.1% to 7.6%. The relative standard deviations for reproducibility (RSDr) ranged from 7.2% to 25.2%. For NIV, the RSDr ranged from 2.0% to 10.7%, and the RSDr ranged from 7.0% to 31.4%. Regardless of sample and detector, the HorRat values for DON and NIV ranged from 0.4 to 1.4. Both LC-UV and LC-MS(/MS) methods were considered to be suitable for application as an official method.     

Determination of Deoxynivalenol and Nivalenol in Wheat by Ultra-Performance Liquid Chromatography/Photodiode-Array Detector and Immunoaffinity Column Cleanup

An ultra-performance liquid chromatography (UPLC®) method has been developed for the simultaneous determination of deoxynivalenol (DON) and nivalenol (NIV) in wheat. Ground sample was extracted with water and the filtered extract was cleaned up through an immunoaffinity column containing a monoclonal antibody specific for DON and NIV. Toxins were separated and quantified by UPLC® with photodiode-array detector (λ?=?220 nm) in less than 3 min. Mean recoveries from blank wheat samples spiked with DON and NIV at levels of 100–2,000 μg/kg (each toxin) ranged from 85 to 95 % for DON and from 81 to 88 % for NIV, with relative standard deviations less than 7 %. Similar recoveries were observed from spiked samples when methanol/water (80:20, v/v) was used as extraction solvent. However, by using a wheat sample naturally contaminated with DON and NIV, the one-way analysis of variance (Student–Newman–Keuls test) between different extraction solvents and modes showed that water extraction provided a significant increase (P?<?0.001) in toxin concentrations (mean values of six replicate analyses) with respect to methanol/water (80:20, v/v). No significant difference was observed between shaking (60 min) and blending (3 min). The limit of detection (LOD) of the method was 30 μg/kg for DON and 20 μg/kg for NIV (signal-to-noise ratio 3:1). The immunoaffinity columns showed saturation of DON/NIV binding sites at levels higher than 2,000 ng in blank wheat extracts spiked with the corresponding amount of mycotoxin, as single mycotoxin or sum of DON and NIV. The range of applicability of the method was from LOD to 4,000 μg/kg, as single mycotoxin or sum of DON and NIV in wheat. The analyses of 20 naturally contaminated wheat samples showed DON contamination in all analyzed samples at level ranging from 30 to 2,700 μg/kg. NIV was detected in two samples at negligible toxin levels (up to 46 μg/kg). This is the first UPLC® method using immunoaffinity column cleanup for the simultaneous and sensitive determination of DON and NIV in wheat.     

Improved methodology for the simultaneous detection of the trichothecene mycotoxins deoxynivalenol and nivalenol in cereals

A simple and sensitive method has been developed for the analysis of two trichothecene mycotoxins (deoxynivalenol and nivalenol) in cereals. These toxins were extracted with acetonitrile/water (3:1), defatted with n-hexane and purified by a two-step chromatographic procedure using Florisil and Sep-pak columns. The amounts of deoxynivalenol (DON) and nivalenol (NIV) in the column eluates were quantitated by gas chromatography with electron capture detector and gas chromatography-mass spectrometry (selected ion monitoring). The limits of detection of the method were 2.0 micrograms/kg for DON and NIV with recoveries of the toxins spiked into polished rice, wheat and corn at 300 micrograms/kg averaging 87% and 86% respectively.     

A survey on the occurrence of Fusarium mycotoxins in Bavarian cereals from the 1987 harvest

Bavarian cereals and wheat flour from the 1987 harvest were analysed for nivalenol (NIV) and deoxynivalenol (DON) using high performance liquid chromatography (HPLC) and for T-2 toxin and zearalenone (ZEA) by enzyme-linked-immunosorbent assay (ELISA). The study included 190 field samples of wheat, barley, rye and oat with visibly damaged ears, 45 samples of wheat intended for feed production and two series of wheat flour (type 550) and whole wheat flour collected in October 1987 and June 1988. The field samples examined showed a high DON contamination of wheat (87%) with an average of 3.96 mg/kg and a maximum of 43.8 mg/kg. Mean levels between 0.33 mg/kg and 0.27 mg/kg DON could be detected in barley, rye and oat. Of the wheat samples, 58% contained ZEA with a maximum of 1.560 mg/kg. The highest levels of ZEA were detected in samples which also showed high concentrations of DON. The NIV and T-2 toxin levels were comparatively low. Thirty percent of the samples showed NIV concentrations between 0.04 mg/kg and 0.29 mg/kg and 38% contained between 0.005 and 0.60 mg/kg of T-2. In the wheat samples for feed production, only DON was detected with an average of 0.190 mg/kg and a maximum of 0.75 mg/kg. The highest DON levels (0.58 mg/kg) from October 1987 were found in the wheat flour samples which were lower than the highest DON concentration (3.24 mg/kg) detected in the samples collected during June 1988. This fact was probably due to a substantial amount of non-contaminated wheat from 1986. The toxin concentrations in the whole wheat flour were not higher than in the type 550 flour.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of Deoxynivalenol in Wheat Bran and Whole-Wheat Flour by Fluorescence Polarization Immunoassay

A rapid and accurate fluorescence polarization (FP) immunoassay has been optimized for the determination of deoxynivalenol (DON) in wheat bran and whole-wheat flour. A preliminary treatment with activated charcoal was used to eliminate the strong matrix effect due to highly colored interfering compounds present in raw wheat bran extracts. In particular, matrix effect was removed by adding activated charcoal to the wheat bran extract (3.5 mg/mL) and mixing for 3 min of incubation time prior to the FP immunoassay analysis. No preliminary treatment was necessary for whole-wheat flour. Average recoveries from samples spiked with DON at levels of 500, 1,000, and 1,500 μg/kg were 95 % for wheat bran and 94 % for whole-wheat flour, with relative standard deviation generally lower than 13 %. Limits of quantification of the optimized FP immunoassay were 120 μg/kg for both matrices. The overall time of analysis was lower than 15 min for wheat bran and 10 min for whole-wheat flour. Good correlations (r?>?0.971) were observed between DON contents obtained by both FP immunoassay and high-performance liquid chromatography with immunoaffinity cleanup for 37 and 23 samples of naturally contaminated wheat bran and whole-wheat flour, respectively. These results show that the FP immunoassay is suitable for high-throughput screening as well as for quantitative determination of DON in wheat bran and whole-wheat flour.     

Evaluation of Dispersive Liquid–Liquid Microextraction–HPLC–UV for Determination of Deoxynivalenol (DON) in Wheat Flour

A fast and simple method for the extraction of deoxynivalenol (DON) from wheat flour using dispersive liquid–liquid microextraction (DLLME) followed by high-performance liquid chromatography–UV detection has been developed and compared with immunoaffinity column cleanup (IAC) process. The influence of several important parameters on the extraction efficacy was studied. Under optimized conditions, a linear calibration curve was obtained in the range of 50–1,000 μg/L. Average recoveries of DON from spiked wheat samples at levels of 500 μg/kg for DLLME and IAC ranged from 72.9?±?1.6 and 85.5?±?3.1, respectively. A good correlation was found for spiked samples between DLLME and IAC methods. The limit of detection was 125 and 50 μg/kg for DLLME and IAC method, respectively. Advantages of DLLME method with respect to the IAC have been pointed out.     

A survey on the occurrence of Fusarium mycotoxins in Bavarian cereals from the 1987 harvest

Summary Bavarian cereals and wheat flour from the 1987 harvest were analysed for nivalenol (NIV) and deoxynivalenol (DON) using high performance liquid chromatography (HPLC) and for T-2 toxin and zearalenone (ZEA) by enzyme-linked-immunosorbent as say (ELISA). The study included 190 field samples of wheat, barley, rye and oat with visibly damaged ears, 45 samples of wheat intended for feed production and two series of wheat flour (type 550) and whole wheat flour collected in October 1987 and June 1988. The field samples examined showed a high DON contamination of wheat (87%) with an average of 3.96 mg/kg and a maximum of 43.8 mg/kg. Mean levels between 0.33 mg/kg and 0.27 mg/kg DON could be detected in barley, rye and oat. Of the wheat samples, 58% contained ZEA with a maximum of 1.560 mg/kg. The highest levels of ZEA were detected in samples which also showed high concentrations of DON. The NIV and T-2 toxin levels were comparatively low. Thirty percent of the samples showed NIV concentrations between 0.04 mg/kg and 0.29 mg/kg and 38% contained between 0.005 and 0.60 mg/kg of T-2. In the wheat samples for feed production, only DON was detected with an average of 0.190 mg/kg and a maximum of 0.75 mg/kg. The highest DON levels (0.58 mg/kg) from October 1987 were found in the wheat flour samples which were lower than the highest DON concentration (3.24 mg/kg) detected in the samples collected during June 1988. This fact was probably due to a substantial amount of non-contaminated wheat from 1986. The toxin concentrations in the whole wheat flour were not higher than in the type 550 flour. The regional distribution of the mean DON concentrations showed the highest levels in Middle and Lower-Franconia.

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Vorkommen von Fusarium Mykotoxinen in bayerischem Getreide der Ernte 1987

Zusammenfassung Cerealien und Weizenmehle der bayerischen Ernte 1987 wurden mittels hochauflösender Flüssigchromatographie (HPLC) auf Nivalenol (NIV) und Deoxynivalenol (DON) Bowie mit Enzymimmunoassay auf T-2 Toxin und Zearalenon (ZEA) analysiert. Die Untersuchungen umfaßten 190 Feldproben von Weizen, Gerste, Roggen und Hafer, die alle optisch erkennbaren Fusarienbefall aufwiesen, 45 Futterweizenproben Bowie zwei Probenserien von Weizenmehlen der Type 550 und Vollkornweizenmehlen, die im October 1987 und im Juni 1988 gezogenwurden. — Die Untersuchungen der Feldproben ergaben eine hohe DON-Kontamination des Weizens (87%) mit einem durchschnittlichen Gehalt von 3,96 mg/kg und einem Maximalgehalt von 43,8 mg/kg. In Gerste, Roggen und Hafer konnten durchschnittlich zwischen 0,33 mg/kg und 0,27 mg/kg DON-nachgewiesen werden. 58% der Winterweizenproben wiesen Zearalenon mit einem Maximalgehalt von 1,56 mg/kg auf. Die höchsten ZEA-Werte wurden in Proben ermittelt, die gleichzeitig einen hohen DON-Gehalt aufwiesen. Die Konzentrationen von NIV und T-2 Toxin waren vergleichsweise niedrig. 30% der Proben hatten NIV-Gehalte zwischen 0,04 mg/kg und 0,29 mg/kg und 38% enthielten T-2 Toxin zwischen 0,005 mg/kg und 0,06 mg/kg. In den Futterweizenproben konnte DON als einziges Toxin mit einem Gehalt von durchschnittlich 0,19 mg/kg und maximal 0,75 mg/kg festgestellt werden. Die Weizenmehle, die im October 1987 gezogen wurden, wiesen maximal 0,58 mg/kg DON auf. Die Gehalte lagen damit medriger als die der Mehlproben vom Juni 1988, die maximal 3,24 mg/kg und durchschnittlich 0,26 mg/kg DON enthielten. Dieser Sachverhalt könnte auf Anteile von nicht kontaminiertem Weizen der Ernte 86 an den im October gezogenen Mehlproben zurückgeführt werden. Die Toxingehalte der Vollkornmehle waren nicht höher als die der Weizenmehle der Type 550. Die höchsten Durchschnittsgehalte von DON wurden in Mittel- und Unterfranken festgestellt.

     

Simultaneous Determination of Deoxynivalenol,Deoxynivalenol-3-Glucoside and Nivalenol in Wheat Grains by HPLC-PDA with Immunoaffinity Column Cleanup

Deoxynivalenol-3-glucoside (D3G) is a modified mycotoxin formed by the metabolism of plants through the conjugation of deoxynivalenol (DON) with glucose. Toxicology studies of D3G for human and animal health are still under investigation, and the development of practical and reliable methods for its direct determination, especially in cereal matrices, is of great importance. In the present study, a methodology for simultaneous determination of D3G, DON, and nivalenol (NIV) in wheat grains, using immunoaffinity column (IAC) cleanup, separation by C18 column and detection by ultraviolet (UV) absorption, was optimized and in-house validated. The results demonstrated adequate values of D3G recovery from IAC and spiked samples. Intraday precision, linearity, limit of detection and limit of quantification (LOQ) were also adequate for the determination of these mycotoxins. Range of applicability varied from 47.1 to 1000 μg/kg for D3G and from 31.3 to 1000 μg/kg for DON and NIV, with recovery ranging from 84.7?±?7.2 % to 112.3?±?8.1 %. A high incidence of D3G (41.2 %, all samples <LOQ) was verified in commercial samples of wheat grains and whole wheat-flour (n?=?17). Also, the presence of D3G occurred simultaneously with DON in 100 % of the D3G-positive samples. DON levels varied from <LOQ to 325.8 μg/kg, and NIV was detected in only 29.5 % (from <LOQ to 140.6 μg/kg). To the best of our knowledge, this is the first method of simultaneous determination of NIV, D3G, and DON by high-performance liquid chromatography with photodiode array detector (HPLC-PDA) reported until now.     

Optimization of a fluorescence polarization immunoassay for rapid quantification of deoxynivalenol in durum wheat-based products

A fluorescence polarization immunoassay previously described for deoxynivalenol (DON) screening in wheat was optimized for the rapid quantification of DON in durum wheat kernels, semolina, and pasta. A background signal was observed in both spiked and naturally contaminated samples, strictly depending on the testing matrix. After subtracting the background DON level for durum wheat (0.27 microg of DON per g), semolina (0.08 microg of DON per g), and pasta (0.04 microg of DON per g), an accurate quantification of DON was possible at levels greater than 0.10 microg/g for all matrices. Average recoveries from spiked samples (0.25 to 1.75 microg/g) were 98, 102, and 101% for wheat, semolina, and pasta, respectively. Comparative analyses of 35 naturally contaminated durum wheat samples, 22 semolina samples, and 26 pasta samples performed by both the fluorescence polarization method and high-pressure liquid chromatography/immunoaffinity cleanup showed a good correlation (r > 0.995). The fluorescence polarization method showed better accuracy and precision with respect to the high-pressure liquid chromatography method and is suitable for the rapid and quantitative determination of DON in durum wheat-based products at levels foreseen by existing or coming international regulations.     

毛细管气相色谱法测定小麦和玉米中脱氧雪腐镰刀菌烯醇

本文采用毛细管气相色谱法测定了小麦和玉米中脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)的含量,小麦、玉米样品分别用乙腈-水(84:16,V/V)提取,小麦样品提取液用硅镁型吸附剂净化,玉米样品提取液用硅镁型吸附剂和活性炭二步净化,采用N-七氟丁酰咪唑(HFBI)为衍生剂,带电子捕获检测器的GC进行检测。DON加标量为0．5～1．5mg/kg时,5次重复实验的平均回收率:小麦样品为82．2%～98．5%,玉米样品为86．0%～103．4%,相对标准偏差(RSD)分别为6．2%～7．6%和6．4%～8．0%,该法DON的最低检出限为0．01mg/kg,线性范围为0．075～15mg/k8。用该法对21个小麦样品与20个玉米样品中DON的含量进行检测,其中小麦样品中DON含量为0．153～4．618mg/kg,污染率为47．6%,超标率为9．5%;而玉米样品中DON含量为0．116～3．004mg/kg,污染率为85%,超标率为20%。