Journal of Integrative Agriculture ›› 2021, Vol. 20 ›› Issue (11): 2966-2975.DOI: 10.1016/S2095-3119(20)63492-X

所属专题： 植物病理合辑Plant Protection—Plant Pathology； 植物病毒合辑Plant Virus

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  • 收稿日期:2020-06-03 出版日期:2021-11-01 发布日期:2021-09-17

Three sensitive and reliable serological assays for detection of potato virus A in potato plants

WU Jia-yu1*, ZHANG Yu2*, ZHOU Xue-ping2, 3, QIAN Ya-juan1, 2 
  

  1. 1 Department of Applied Biological Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, P.R.China
    2 State Key Laboratory of Rice Biology, Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, P.R.China
    3 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China 
  • Received:2020-06-03 Online:2021-11-01 Published:2021-09-17
  • Contact: Correspondence QIAN Ya-juan, Tel: +86-571-88982677, E-mail: yjqian@zju.edu.cn
  • About author: WU Jia-yu, E-mail: 1239324936@qq.com; * These authors contributed equally to this study.
  • Supported by:
    This work was supported by the National Key Research and Development Program of China (2017YFD0201604) and the National Natural Science Foundation of China (31571976).

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摘要：

马铃薯A病毒（potato virus A，PVA）是马铃薯种植区域侵染马铃薯的常见且经济重要的病毒之一，建立快速、灵敏、高通量的PVA检测技术对防控该病毒病害具有重要意义。本研究利用感染PVA的马铃薯植物中提纯的PVA病毒粒子为免疫原，免疫BALB/c小鼠，经杂交瘤技术获得4株能分泌抗PVA单克隆抗体的杂交瘤细胞株(2D4、8E11、14A6和16H10)。Western blot分析发现，4株单抗均与PVA的假定外壳蛋白亚基有特异性免疫反应。以4株杂交瘤细胞分泌的单抗为核心相继建立了检测马铃薯叶片和马铃薯块茎等中PVA的抗原包被板子酶联免疫吸附试验（antigen-coated plate enzyme-linked immunosorbent assay，ACP-ELISA）、斑点酶联免疫吸附试验（Dot-ELISA）和组织印迹酶联免疫吸附试验（Tissue print-ELISA）三种血清学方法。特异性分析结果表明，ACP-ELISA和Dot-ELISA检测感染PVA马铃薯病叶的灵敏度达到1:327680倍和1:10240倍稀释 (w/v, g/mL)。Tissue print-ELISA是这三种血清学检测方法中最快、最简便的检测技术，更适合现场大规模样品检测。利用建立的三种血清学方法对2019年从云南省和浙江省田间采集的22个马铃薯样品进行检测，发现有3个样品感染PVA。进一步通过RT-PCR检测和PVA CP基因的克隆测序及核酸序列比对证实3种PVA血清学检测方法在马铃薯田间样品调查上的准确性。我们的研究结果表明，灵敏、特异PVA单抗的创制及血清学检测方法的建立在PVA田间检测、病害流行病学的研究、马铃薯无毒种薯的生产方面具有巨大应用潜力。


Abstract:

Vegetative propagation of seed potato often allows passaging of viruses to seed tubers, resulting in significant yield losses and reduction of potato tuber quality.  Thus, virus detection approach is crucial for effective virus management programs and the production of virus-free seed potatoes.  Among the reported potato-infecting viruses, potato virus A (PVA) is considered as one of the most important viruses in potato-growing regions worldwide.  This study prepared four hybridoma lines secreting PVA-specific monoclonal antibodies (MAbs) (2D4, 8E11, 14A6 and 16H10) using purified PVA virions as an immunogen.  Western blotting results indicated that all the four MAbs reacted strongly and specifically with the putative capsid protein of PVA.  Using these four MAbs, this study developed antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA), Dot-ELISA and Tissue print-ELISA for detection of PVA infection in potato plants.  The results indicated that PVA can be detected in crude tissue extracts from infected potato plants diluted up to 1:327 680 (w/v, g mL–1) by ACP-ELISA or up to 1:10 240 by Dot-ELISA.  The Tissue print-ELISA is the quickest and easiest approach among the three serological assays, and is more suitable for onsite large-scale potato screening programs.  Further analyses of field-collected potato samples showed that the sensitivities and specificities of the three serological approaches were similar to those of RT-PCR in PVA detection and confirmed that PVA is currently widespread in Yunnan and Zhejiang provinces of China.  Hence, the results strongly suggest that these highly sensitive serological approaches based on PVA-specific MAbs are useful and powerful for PVA-free seed potato production programs and PVA field surveys. 

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