Aphid-borne Zucchini yellow mosaic virus （ZYMV） is one of the most economically important viruses of cucurbitaceous plants. To survey and control this virus, it is necessary to develop an efficient detection techniq...Aphid-borne Zucchini yellow mosaic virus （ZYMV） is one of the most economically important viruses of cucurbitaceous plants. To survey and control this virus, it is necessary to develop an efficient detection technique. Using purified ZYMV virion and the conventional hybridoma technology, three hybridoma cell lines （16A11, 5A7 and 3B8） secreting monoclonal antibodies （MAbs） against ZYMV Zhejiang isolate were obtained. The working titers of the ascitic fluids secreted by the three hybridoma cell lines were up to 10^-7 by indirect enzyme-linked immunosorbent assay （ELISA）. All MAbs were isotyped as IgG1, kappa light chain. Western blot analysis indicated that the MAb 3B8 could specifically react with the coat protein of ZYMV while MAbs 5A7 and 16A11 reacted strongly with a protein of approximately 51 kDa from the ZYMV-infected leaf tissues. According to this molecular weight, we consider this reactive protein As likely to be the HC-Pro protein. Using these three MAbs, we have now developed five detection assays, i.e., antigen-coated-plate ELISA （ACP-ELISA）, dot-ELISA, tissue blot-ELISA, double-antibody sandwich ELISA （DAS-ELISA）, and immunocapture-RT-PCR （IC-RT-PCR）, for the sensitive, specific, and easy detection of ZYMV. The sensitivity test revealed that ZYMV could be readily detected respectively by ACP-ELISA, dot-ELISA, DAS-ELISA and IC-RT-PCR in 1：163840, 1：2560, 1：327680 and 1：1 310720 （w/v, g mL-1） diluted crude extracts from the ZYMV-infected plants. We demonstrated in this study that the dot-ELISA could also be used to detect ZYMV in individual viruliferous aphids. A total of 275 cucurbitaceous plant samples collected from the Zhejiang, Jiangsu, Shandong and Hainan provinces, China, were screened for the presence of ZYMV with the described assays. Our results showed that 163 of the 275 samples （59%） were infected with ZYMV. This finding indicates that ZYMV As now widely present in cucurbitaceous crops in China. RT-PCR followed by DNA sequencing and sequence analyses confirmed the accuracy of the five assays. We consider that these detection assays can significantly benefit the control of ZYMV in China.展开更多
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（...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-C2 X 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, 1：1 280 and 1：655 360（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 1：12 800 and 1：1 600（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.展开更多
基金supported by the National Natural Science Foundation of China(31272015)the National Basic Research Program(973)of China(2014CB138400)the Special Fund for Agro-scientific Research in the Public Interest,China(201303021,201303028)
文摘Aphid-borne Zucchini yellow mosaic virus （ZYMV） is one of the most economically important viruses of cucurbitaceous plants. To survey and control this virus, it is necessary to develop an efficient detection technique. Using purified ZYMV virion and the conventional hybridoma technology, three hybridoma cell lines （16A11, 5A7 and 3B8） secreting monoclonal antibodies （MAbs） against ZYMV Zhejiang isolate were obtained. The working titers of the ascitic fluids secreted by the three hybridoma cell lines were up to 10^-7 by indirect enzyme-linked immunosorbent assay （ELISA）. All MAbs were isotyped as IgG1, kappa light chain. Western blot analysis indicated that the MAb 3B8 could specifically react with the coat protein of ZYMV while MAbs 5A7 and 16A11 reacted strongly with a protein of approximately 51 kDa from the ZYMV-infected leaf tissues. According to this molecular weight, we consider this reactive protein As likely to be the HC-Pro protein. Using these three MAbs, we have now developed five detection assays, i.e., antigen-coated-plate ELISA （ACP-ELISA）, dot-ELISA, tissue blot-ELISA, double-antibody sandwich ELISA （DAS-ELISA）, and immunocapture-RT-PCR （IC-RT-PCR）, for the sensitive, specific, and easy detection of ZYMV. The sensitivity test revealed that ZYMV could be readily detected respectively by ACP-ELISA, dot-ELISA, DAS-ELISA and IC-RT-PCR in 1：163840, 1：2560, 1：327680 and 1：1 310720 （w/v, g mL-1） diluted crude extracts from the ZYMV-infected plants. We demonstrated in this study that the dot-ELISA could also be used to detect ZYMV in individual viruliferous aphids. A total of 275 cucurbitaceous plant samples collected from the Zhejiang, Jiangsu, Shandong and Hainan provinces, China, were screened for the presence of ZYMV with the described assays. Our results showed that 163 of the 275 samples （59%） were infected with ZYMV. This finding indicates that ZYMV As now widely present in cucurbitaceous crops in China. RT-PCR followed by DNA sequencing and sequence analyses confirmed the accuracy of the five assays. We consider that these detection assays can significantly benefit the control of ZYMV in China.
基金supported by the National Basic Research Program of China(2010CB126203)the special fund for Agro-scientific Research in the Public Interest,China(201003031)+1 种基金Earmarked Funds for Modern Agro-industry Technology Research SystemZhejiang Provincial Natural Science Foundation of China(Z3090039)
文摘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-C2 X 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, 1：1 280 and 1：655 360（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 1：12 800 and 1：1 600（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.
