Multiplex detection of plant pathogens using a microsphere immunoassay technology.
Multiplex detection of plant pathogens using a microsphere immunoassay technology.
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DOI：
10.1371/journal.pone.0062344
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Elliott CT
中科院分区：
文献类型：
--
作者：
Charlermroj R;Himananto O;Seepiban C;Kumpoosiri M;Warin N;Oplatowska M;Gajanandana O;Grant IR;Karoonuthaisiri N;Elliott CT
植物病原体是种子出口、植物病害控制和植物检疫的一个严重问题。迫切需要快速、准确的筛选测试来保护和防止植物病害在全球范围内传播。基于微球免疫分析开发了一种新型多重检测方法，可同时检测四种重要的植物病原体：果斑病菌燕麦酸食亚种（Acidovorax avenae subsp.）。西瓜病毒（Aac）、辣椒脉带斑驳病毒（CVbMV，potyvirus）、西瓜银斑驳病毒（WSMoV，tospovirus 血清群 IV）和瓜黄斑病毒（MYSV，tospovirus）。每种植物病原体的抗体都连接在荧光编码的磁性微球组上，用于捕获相应的病原体。通过 R-藻红蛋白 (RPE) 标记的病原体特异性抗体检测病原体的存在。通过确定合适的抗体对、封闭缓冲液、RPE 标记抗体的浓度和测定时间来优化测定条件。一旦条件得到优化，当加入缓冲液和健康西瓜叶提取物时，该测定能够精确地检测所有四种植物病原体，其灵敏度比酶联免疫吸附测定 (ELISA) 高得多。微球免疫分析的分析时间（1小时）比ELISA的分析时间（4小时）短得多。该系统还被证明能够检测自然感染的植物样品中的病原体，是植物病原体检测的重大进步。
Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV, potyvirus), watermelon silver mottle virus (WSMoV, tospovirus serogroup IV) and melon yellow spot virus (MYSV, tospovirus). An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE)-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour) was much shorter than that of ELISA (4 hours). This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection.