Highly Sensitive and Specific Monoclonal Antibody-Based Serological Methods for Rice Ragged Stunt Virus Detection in Rice Plants and Rice Brown Planthopper Vectors

doi:10.1016/S2095-3119(13)60533-X

Journal of Integrative Agriculture ›› 2014, Vol. 13 ›› Issue (9): 1943-1951.DOI: 10.1016/S2095-3119(13)60533-X

Highly Sensitive and Specific Monoclonal Antibody-Based Serological Methods for Rice Ragged Stunt Virus Detection in Rice Plants and Rice Brown Planthopper Vectors

LIU Huan, SONG Xi-jiao, NI Yue-qun, LU Li-na, ZHOU Xue-ping , WU Jian-xiang

Institute of Biotechnology, College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310058, P.R.China

收稿日期:2013-03-28 出版日期:2014-09-22 发布日期:2014-09-24
通讯作者: WU Jian-xiang, Tel: +86-571-88982250, Fax: +86-571-86971498, E-mail: wujx@zju.edu.cn
作者简介:LIU Huan, E-mail: liuh2010sjz@163.com
基金资助:
This work was supported by the National Basic Research Program of China (2010CB126203), the special fund for Agro- scientific Research in the Public Interest, China (201003031), and Earmarked Funds for Modern Agro-industry Technology Research System and Zhejiang Provincial Natural Science Foundation of China (Z3090039).

Highly Sensitive and Specific Monoclonal Antibody-Based Serological Methods for Rice Ragged Stunt Virus Detection in Rice Plants and Rice Brown Planthopper Vectors

LIU Huan, SONG Xi-jiao, NI Yue-qun, LU Li-na, ZHOU Xue-ping , WU Jian-xiang

Institute of Biotechnology, College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310058, P.R.China

Received:2013-03-28 Online:2014-09-22 Published:2014-09-24
Contact: WU Jian-xiang, Tel: +86-571-88982250, Fax: +86-571-86971498, E-mail: wujx@zju.edu.cn
About author:LIU Huan, E-mail: liuh2010sjz@163.com
Supported by:
This work was supported by the National Basic Research Program of China (2010CB126203), the special fund for Agro- scientific Research in the Public Interest, China (201003031), and Earmarked Funds for Modern Agro-industry Technology Research System and Zhejiang Provincial Natural Science Foundation of China (Z3090039).

摘要/Abstract

摘要： Rice ragged stunt virus (RRSV) is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein (CP) gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21 (DE3) using the pMAL-C2X expression vector. The recombinant protein was used as the immunogen to immunize BALB/c mice. A hybridoma cell line 8A12 secreting monoclonal antibody (MAb) against RRSV was obtained by fusing mouse myeloma cells (Sp 2/0) with spleen cells from the immunized BALB/c mice. Western blot analysis showed that the MAb 8A12 can specifically react with RRSV CP. Using the MAb, an antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA), a dot enzyme-linked immunosorbent assay (dot-ELISA), and immunocapture-RT-PCR (IC-RT-PCR) assay were developed to detect RRSV. The established ACP-ELISA, dot-blot ELISA and IC-RT-PCR methods could detect RRSV in infected rice tissue crude extracts with dilutions of 1:40 960, 1:1280 and 1:655360 (w/v, g mL-1), respectively. The ACP-ELISA and dot-blot ELISA methods could detect RRSV in infected insect vector crude extracts with dilutions of 1:12800 and 1:1600 (an individual planthopper μL-1), respectively. The field survey revealed that Rice ragged stunt disease occurs on rice in Hainan, Yunnan, Guangxi, Sichuan, Guizhou, Fujian, Hunan, Jiangxi and Zhejiang in China.

关键词: Rice ragged stunt virus , rice brown planthopper , monoclonal antibody , antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA) , dot-blot ELISA , immunocapture RT-PCR

Abstract: Rice ragged stunt virus (RRSV) is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein (CP) gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21 (DE3) using the pMAL-C2X expression vector. The recombinant protein was used as the immunogen to immunize BALB/c mice. A hybridoma cell line 8A12 secreting monoclonal antibody (MAb) against RRSV was obtained by fusing mouse myeloma cells (Sp 2/0) with spleen cells from the immunized BALB/c mice. Western blot analysis showed that the MAb 8A12 can specifically react with RRSV CP. Using the MAb, an antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA), a dot enzyme-linked immunosorbent assay (dot-ELISA), and immunocapture-RT-PCR (IC-RT-PCR) assay were developed to detect RRSV. The established ACP-ELISA, dot-blot ELISA and IC-RT-PCR methods could detect RRSV in infected rice tissue crude extracts with dilutions of 1:40 960, 1:1280 and 1:655360 (w/v, g mL-1), respectively. The ACP-ELISA and dot-blot ELISA methods could detect RRSV in infected insect vector crude extracts with dilutions of 1:12800 and 1:1600 (an individual planthopper μL-1), respectively. The field survey revealed that Rice ragged stunt disease occurs on rice in Hainan, Yunnan, Guangxi, Sichuan, Guizhou, Fujian, Hunan, Jiangxi and Zhejiang in China.

Key words: Rice ragged stunt virus , rice brown planthopper , monoclonal antibody , antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA) , dot-blot ELISA , immunocapture RT-PCR

LIU Huan, SONG Xi-jiao, NI Yue-qun, LU Li-na, ZHOU Xue-ping , WU Jian-xiang. Highly Sensitive and Specific Monoclonal Antibody-Based Serological Methods for Rice Ragged Stunt Virus Detection in Rice Plants and Rice Brown Planthopper Vectors[J]. Journal of Integrative Agriculture, 2014, 13(9): 1943-1951.

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Highly Sensitive and Specific Monoclonal Antibody-Based Serological Methods for Rice Ragged Stunt Virus Detection in Rice Plants and Rice Brown Planthopper Vectors

Highly Sensitive and Specific Monoclonal Antibody-Based Serological Methods for Rice Ragged Stunt Virus Detection in Rice Plants and Rice Brown Planthopper Vectors

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