食品科学 ›› 2019, Vol. 40 ›› Issue (8): 256-261.doi: 10.7506/spkx1002-6630-20171128-337

• 安全检测 • 上一篇    下一篇

基于噬菌体展示纳米抗体的绿色免疫PCR检测脱氧雪腐镰刀菌烯醇

江东健,罗秀儿,何庆华   

  1. 1.南昌大学 食品科学与技术国家重点实验室,中德联合研究院,江西 南昌 330047;2.南昌大学食品学院,江西 南昌 330031
  • 出版日期:2019-04-25 发布日期:2019-05-05
  • 基金资助:
    国家自然科学基金面上项目(NSFC-31671924);国家自然科学基金地区科学基金项目(NSFC-31360386);江西省杰出青年人才资助计划项目(20171BCB23023);江西省重大科技项目(20143ACB21008)

Phage Displayed Nanobody Mediated Green Immuno-PCR for Detection of Deoxynivalenol

JIANG Dongjian, LUO Xiu’er, HE Qinghua   

  1. 1. State Key Laboratory of Food Science and Technology, Sino German Joint Research Institute, Nanchang University, Nanchang 330047, China; 2. School of Food Science & Technology, Nanchang University, Nanchang 330031, China
  • Online:2019-04-25 Published:2019-05-05

摘要: 目的:在获得脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)抗独特型纳米抗体的前期基础上,将纳米抗体作为酶标抗原的替代物,应用于荧光定量免疫聚合酶链式反应(polymerase chain reaction,PCR)体系,实现DON的高灵敏、绿色免疫分析。方法:将特异性结合DON抗体的噬菌体展示纳米抗体(P-28)作为竞争抗原,以编码P-28纳米抗体的DNA为靶标,设计特异性PCR扩增引物,优化荧光定量免疫PCR退火温度、抗DON抗体浓度、P-28投入量等参数,建立基于间接竞争模式的荧光定量免疫PCR检测DON的方法。结果:本研究建立的荧光定量免疫PCR方法线性检测范围为0.1~1 000 ng/mL,IC50值为(3.96±2.21)ng/mL,最低检出限为0.048 ng/mL,并与其他真菌毒素无交叉反应。结论:该方法直接使用噬菌体展示纳米抗体作为竞争抗原的替代物,应用于免疫PCR体系,避免了使用传统的化学合成酶标抗原所带来的环境污染、操作毒性等缺陷,并具有良好的特异性及灵敏度。

关键词: 脱氧雪腐镰刀菌烯醇, 抗独特性抗体, 噬菌体展示纳米抗体, 酶联免疫吸附测定, 免疫PCR

Abstract: Objective: In order to develop a highly sensitive and green immunoassay for deoxynivalenol (DON) anti-idiotypic nanobody, obtained in our previous study, was used as an alternative to enzyme-labeled antigen in a fluorescence real-time immuno-PCR system. Methods: The phage displayed nanobody (P-28), which specifically binds with anti-DON antibody, was used as a competitive antigen and the DNA encoding P-28 Nanobody was used as a target for primer design. Important experimental parameters including annealing temperature, anti-DON antibody concentration and P-28 amount were optimized. Finally, we proposed a fluorescence real-time immuno-PCR method based on indirect competition for the detection of DON. Results: The linear range of the immuno-fluorescence PCR method was 0.1–1 000 ng/mL with an IC50 of (3.96 ± 2.21) ng/mL, and the limit of detection (LOD) was 0.048 ng/mL. This method showed no crossreaction with other mycotoxins. Conclusion: This method avoids the defects of environmental pollution and operational toxicity caused by using traditional synthetic enzyme-labelled antigen and has good specificity and sensitivity.

Key words: deoxynivalenol, anti-idiotypic antibody, phage displayed nanobody, enzyme-linked immunosorbent assay, immuno-PCR

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