【作者】 尹珺；

【导师】 张海彬；

【作者基本信息】 南京农业大学 ， 临床兽医学， 2006， 硕士

【摘要】 脱氧雪腐镰刀菌烯醇（DON）是镰刀菌产生的一种真菌毒素，主要污染谷物及其制品。食用污染毒素的谷物，如病麦及其制成品后会引起人畜中毒，还有致癌、致畸和诱变的作用。谷物往往是通过产毒真菌的感染而污染毒素的，如小麦赤霉病，是影响我国南方麦区、东北春麦区以及黄淮流域麦区的重要病害，可由多种镰刀菌引起，但主要致病种是禾谷镰刀菌（Fusarium graminearum）。镰刀菌引起小麦的穗腐，不仅使小麦籽粒产量降低、品质变劣，而且病麦粒中存留有病菌产生的真菌毒素，如脱氧雪腐镰刀菌烯醇（Deoxynivalenol，DON），但是我国相关的研究比较少，特别是检测水平偏低。因此，对我国禾谷镰刀菌产毒特点进行分析，并改进毒素检测方法，对我国麦制品中真菌毒素含量进行日常化的和标准化的检测，并发布相关毒素污染情况是非常必要的。 本论文对DON的快速检测进行了研究，建立并比较了理化检测方法和免疫学检测方法，主要研究内容和结果如下： 1、为了获得大量DON，将禾谷镰刀菌菌株进行了产毒培养。结果发现，禾谷镰刀菌在约含25％水的小麦培养基中，25℃，振荡培养30天，产毒量最高； 2、DON的提取试验结果表明，小麦样品宜用乙腈-水（84：16，V／V）提取，硅镁型吸附剂柱净化； 3、建立了薄层层析（TLC）检测DON的方法：以展开剂氯仿：甲醇（90：10）展开DON毒素的Rf值约为0.45，DON在365nm紫外光下显蓝色荧光，经20％硫酸喷雾并加热后，DON样点呈玫瑰红或玫瑰紫色； 4、建立了高效液相色谱紫外分析（HPLC-UV）检测DON的方法：以kromasil C-18分析柱（4.6×250mm）为固定相，乙腈-水（84：16）为流动相，流速为0.8mμL／min，进样量为20μL，紫外检测波长为218nm，毒素DON的检测谱峰峰形清晰明显，无干扰，保留时间为8分钟左右。该法最低检出限为0.1ng／μL，线性范围为0.1-10ng／μL； 5、建立了气相色谱-质谱联用（GC-MS）检测DON的方法：毒素经C₁₈／Al₂O₃柱（1／3）滤去色素，用三甲基硅烷基衍生剂TMS衍生化，然后以DB-5MS石英交联毛细管柱（30cm×0.25mm×0.25μm）为固相，纯氦气为载气，流速为1mL／min进行气相色谱；以EI为离子源，扫描间歇1.0s进行质谱分析，特异性好，结果稳定，而且用GC-MS与HPLC检测DON浓度误差不显著； 6、建立了薄层层析（TLC）的定性检测及高效液相色谱（HPLC）的定量检测相结更多还原

【Abstract】 1 Fusarium graminearum was cultured in order to obtain a great deal of DON. The most suitable condition for Fusarium graminearum producing DON is that Fusarium graminearum was cultured in wheat medium for thirty days under 25℃, which contains 15% water.2 Samples were extracted with acetronile-water (84:16,V/V), then purified by a Florisil column.3 The TLC method was established for DON as follows:The Rf value of TLC for DON was 0.45 with chloroform- methanol (90:10,V/V) as expanded matter, and DON sample showed blue fluorescence under 365 nm ultraviolet light or rose-bengal after treated by 20% sulphoric acid.4 The HPLC method for DON was established as follows: Kromasil C-18 column (4.6x250mm) as fixed phase, acetonitrile-water (84:16) as mobile phase,flow rate as 0.8mμL/min, detection sample volumn as 20μL and detection wave as 218nm. Under these conditions, the DON detection peak was obviously clear without disturbance peaks and the retention time was about 8 min. The limit of detection (LOD) of this method was 0.1 ng/μL for DON. Samples with DON levels from 0.1 to 10 ng/μL could be quantitated by this method.5 The GC-MS method was established to detect DON. The toxin was filtered to remove pigment, The final extract was then derivated with TMS and quantitated by GC /MS. The result of this method was comparable with HPLC and ELISA.6 TLC and HPLC were combined together to analyze DON. Firstly, materials containing DON was detected by TLC. And then the band showed blue fluorescence under 365 nm ultraviolet light was collected and analyzed by HPLC to determine the amount and distribution of DON. This method may supply a reference for detection of other mycotoxins.7 The complete antigen was composed as follows: DON obtained a carboxyl after deriving, and then complete antigen of DON (3-HS-DON-BSA or 3-HS-DON-OVA) was更多还原