《Highly Sensitive and Speciifc Monoclonal AntibodyBased Serological Methods for Rice Ragged Stunt Virus Detection in Rice Plants and Rice Brown Planthopper Vectors》.pdfVIP

Journal of Integrative Agriculture 2014, 13(9): 1943-1951 September 2014 RESEARCH ARTICLE 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 and WU Jian-xiang Institute of Biotechnology, College of Agriculture Biotechnology, Zhejiang University, Hangzhou 310058, P.R.China 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 1:1280 and 1:655 360 (w/v, g mL ), respectively. The ACP-ELISA and dot-blot ELISA methods could detect RRSV in infected -1 insect vector crude extracts with dilutions of 1:12 800 and 1:1600 (an individual planthopper µL ), respectively. The field survey revealed that Rice rag