Development and use of three monoclonal antibodies for the detection of rice black-streaked dwarf virus in field plants and planthopper vectors.
Development and use of three monoclonal antibodies for the detection of rice black-streaked dwarf virus in field plants and planthopper vectors.
复制标题
三种单克隆抗体的开发和使用，用于检测田间植物和飞虱媒介中的水稻黑条矮缩病毒
DOI：
10.1186/1743-422x-10-114
复制
发表时间：
2013-04-10
期刊：
影响因子：
4.8
通讯作者：
Zhou X
中科院分区：
文献类型：
--
作者：
Wu J;Ni Y;Liu H;Rao L;Zhou Y;Zhou X
水稻黑条矮缩病毒（RBSDV）在亚洲国家的水稻、玉米和小麦生产中造成了巨大的损失。RBSDV检测的血清学方法的使用取决于抗体的可用性。本研究以RBSDV感染玉米的肿瘤粗提物为免疫原，制备了三种高灵敏度和特异性的抗RBSDV抗原的鼠源单克隆抗体，抗原包被板酶联免疫吸附测定（ACP-ELISA）和斑点酶联免疫吸附试验结果3株单抗均能与RBSDV侵染植物和稻飞虱组织粗提物发生特异性反应。三种单克隆抗体的检测终点ACP-ELISA检测12 E10、18 F10和5G 5的特异性分别为1：40，960、1：40，960和1：40，960。八万一千九百二十（w/v，g mL-1）与感染玉米的粗提物，1：10，240、1：20，480、1：10，240、1：10，240、1：10，480、1：10，480、1：10，48 @@二万零四百八十（w/v，g mL-1）与病株粗提物的比例分别为1：5，120、1：10，240、1：10，240（w/v，g mL-1），与稻飞虱粗提物的比为1：9，600、1：9，600、19，200（个体/μL）。新建立的ACP-ELISA方法可检测稀释度分别为1：81，920、1：20，480、1：10，240（w/v，g mL-1）的玉米、小麦、水稻组织粗提液和稀释度为1：19200（个体/μL）的稻飞虱提取液中的病毒。结果表明，Dot-ELISA能检测到1：320（w/v，g mL-1）稀释的玉米、小麦和水稻组织粗提液中的病毒，也能检测到1：1，600（个体/μL）稀释的稻飞虱组织粗提液中的病毒。应用这两种血清学方法对我国5个省的915份田间植物和594份稻飞虱样品进行了RBSDV的检测。结果表明，915份植物样品中有338份被RBSDV感染，594份稻飞虱样品中有19份被RBSDV感染。田间调查表明，RBSDV在江苏、浙江、山东等省的水稻、玉米和小麦上广泛分布。
BackgroundRice black-streaked dwarf virus (RBSDV) causes great losses in rice, maize and wheat production in Asian countries. The use of serological methods for RBSDV detection depends on the availability of antibodies. In this study, three highly sensitive and specific murine monoclonal antibodies (MAbs) against RBSDV antigens were produced using crude extracts from tumors of RBSDV-infected maize as the immunogen, and two serological assays, antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA) and dot enzyme-linked immunosorbent assay (dot-ELISA) were developed for RBSDV detection.ResultsAll three MAbs reacted strongly and specifically with the crude extracts from RBSDV-infected plant and planthopper tissues. The detection endpoints of three MAbs (12E10, 18F10 and 5G5) in ACP-ELISA were respectively 1:40,960, 1:40,960, 1:81,920 (w/v, g mL-1) with the crude extract of infected maize, 1:10,240, 1:20,480, 1:20,480 (w/v, g mL-1) with the crude extract of infected rice, 1:5,120, 1:10,240, 1:10,240 (w/v, g mL-1) with the crude extract of infected wheat, 1:9,600, 1:9,600, 19,200 (individual planthopper/μL) with the crude extract of infected planthopper. The newly developed ACP-ELISA could detect the virus in the infected maize, wheat, rice tissue crude extracts diluted at 1:81,920, 1:20,480, 1:10,240 (w/v, g mL-1), respectively, and in individual viruliferous planthopper extract diluted at 1:19200 (individual planthopper/μL). The dot-ELISA was proved to detect the virus in the infected maize, wheat and rice tissue crude extracts diluted at 1:320 (w/v, g mL-1), and in individual viruliferous planthopper extract diluted at 1:1,600 (individual planthopper/μL), respectively. Field plants (915) and planthopper samples (594) from five provinces of China were screened for the presence of RBSDV using the two developed serological assays. The results indicated that 338 of the 915 plant samples and 19 of the 594 planthopper samples were infected by RBSDV.ConclusionsThe newly developed ACP-ELISA and dot-ELISA were highly sensitive and specific to detect RBSDV in field plant and planthopper samples. The field survey demonstrated that RBSDV is widespread in rice, maize and wheat crops in Jiangsu, Zhejiang, Shandong provinces of China.