动物营养学报 >

2019 , Vol. 31 >Issue 11: 5229 - 5237

DOI: https://doi.org/10.3969/j.issn.1006-267x.2019.11.039

实验方法 Experimental Methods

脱氧雪腐镰刀菌烯醇间接竞争酶联免疫吸附测定试剂盒的研制及初步应用

  • 韩丽 ,
  • 张文举 ,
  • 王自良 ,
  • 李月涛 ,
  • 姜金庆 ,
  • 李任峰 ,
  • 范国英 ,
  • 张海棠 ,
  • 牛琳琳 ,
  • 吕俊美 ,
  • 周叶
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  • 1. 石河子大学动物科技学院, 石河子 832000;
    2. 河南科技学院动物科技学院, 新乡 453003
韩丽(1980-),女,河南长垣人,博士研究生,从事动物营养与饲料科学研究。E-mail:2505980887@qq.com

收稿日期: 2019-05-10

  网络出版日期: 2019-11-19

基金资助

“十二五”国家科技支撑计划项目“激素类药物快速检测技术研究与示范”（2014BAD13B05-01）；新疆生产建设兵团重点领域科技攻关项目（2018AB041）；第八师现代农业科技攻关与成果转化项目（2018NY05）

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Development and Preliminary Application of an Indirect Competitive ELISA Kit for Detecting Deoxynivalenol

  • HAN Li ,
  • ZHANG Wenju ,
  • WANG Ziliang ,
  • LI Yuetao ,
  • JIANG Jinqing ,
  • LI Renfeng ,
  • FAN Guoying ,
  • ZHANG Haitang ,
  • NIU Linlin ,
  • LYU Junmei ,
  • ZHOU Ye
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  • 1. College of Animal Science and Technology, Shihezi University, Shihezi 832000, China;
    2. College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang 453003, China

Received date: 2019-05-10

  Online published: 2019-11-19

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摘要

本试验旨在研制一种自主组装的间接竞争酶联免疫吸附测定（icELISA）试剂盒，用于饲料中脱氧雪腐镰刀菌烯醇（DON）的检测。在制备抗DON单克隆抗体的基础上，建立icELISA标准曲线，并对其检测方法进行优化，然后用于测定试剂盒各项性能，将测定结果与高效液相色谱（HPLC）法测定结果进行比较分析。结果显示：本试验研制的icELISA试剂盒对DON的最低检测浓度为1.147 ng/mL，检测范围为1.50~130.31 ng/mL，半抑制浓度（IC50）为9.84 ng/mL。对小麦、玉米、猪料、鸡料4个样品进行添加回收试验，回收率在77.1%~113.2%，相对标准偏差（RSD）在4.2%~12.6%。并且，该试剂盒具有特异性高、稳定性良好、有效使用寿命长且样品基质干扰可以忽略不计等优点。利用该试剂盒测定20个盲样，其测定结果与HPLC法测定结果一致性良好。上述结果说明本试验研制的DON icELISA试剂盒各项性能符合相关技术要求，对DON检测具有准确可靠、方便快捷、高通量大等特点，可满足市场对DON的快速初筛需求。

关键词： 脱氧雪腐镰刀菌烯醇; icELISA试剂盒; 性能测定; 初步应用

本文引用格式

韩丽 , 张文举 , 王自良 , 李月涛 , 姜金庆 , 李任峰 , 范国英 , 张海棠 , 牛琳琳 , 吕俊美 , 周叶 . 脱氧雪腐镰刀菌烯醇间接竞争酶联免疫吸附测定试剂盒的研制及初步应用[J]. 动物营养学报, 2019 , 31(11) : 5229 -5237 . DOI: 10.3969/j.issn.1006-267x.2019.11.039

Abstract

This study was conducted to develop a self-assembled blocking indirect competitive ELISA (icELISA) kit for the detection of deoxynivalenol in feed. Based on the preparation of anti-DON monoclonal antibodies, we established the standard curve of icELISA and optimized the detection conditions, which was used to examine the performance of the kit, then the determination results were compared with the high-performance liquid chromatography (HPLC) method. The results showed that the minimum detectable concentration of the kit for DON was 1.147 ng/mL, with the detection range of 1.50 to 130.31 ng/mL, and the 50% inhibiting concentration (IC50) of 9.84 ng/mL. The recovery experiment by adding standard samples was carried out for four samples of wheat, corn, pig feed and chicken feed. The recovery was in the range of 77.1% to 113.2%, and the relative standard deviation (RSD) ranged from 4.2% to 12.6%. Besides, the kit had the advantages of high specificity, good stability, long effective life and negligible sample matrix interference. Finally, twenty blind samples were determined by the kit, and the results were in good agreement with the HPLC method. These results indicate that the icELISA kit developed in this experiment has good performance to meet the relevant technical requirements, and with the characteristics of accuracy, reliability, convenience and high throughput screening to detect DON. Therefore, it is suitable for rapid screening of DON for the market.

Key words： deoxynivalenol; icELISA kit; performance measurement; preliminary application

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