By Using the modified method to extract Rice dwarf virus virions from infected rice leaves,a concentration 3.854mg/mL of purified virus virions reached,and the Rabbits was immunized with purified virions,yielding high...By Using the modified method to extract Rice dwarf virus virions from infected rice leaves,a concentration 3.854mg/mL of purified virus virions reached,and the Rabbits was immunized with purified virions,yielding high titer antiserum;1:20480 by the indirect enzyme-linked immunosorbent assay(I-ELISA)test.The antiserum successfully detected three rice samples from Kunming,Yuxi and Luliang by I-ELISA.Immunocapture polymerase chain reaction(IC-PCR),respectively was the utilized to show that serologic and molecular methods were successfully used for detection of Rice dwarf virus.展开更多
Two kinds of methods RT-PCR and IC-PCR were used to detect pathogen of virus disease of pumpkin and the sensitivity of the two methods was compared. The results showed that PRSV-W and CMV were detected in diseased sam...Two kinds of methods RT-PCR and IC-PCR were used to detect pathogen of virus disease of pumpkin and the sensitivity of the two methods was compared. The results showed that PRSV-W and CMV were detected in diseased samples gathered in Yunnan Province, while WMV and CMV were detected in diseased samples gathered in Heilongjiang Province. The sensitivity of RT-PCR is higher than that of IC-PCR, but the effect of IC-PCR in the specialization of bonding reaction and requisition for experiment material is better than that of RT-PCR.展开更多
采用传统生物学方法、显色培养基方法、自制三抗体夹心ELISA试剂盒(TAS-ELISA)、基因组直接PCR(DNA Direct-PCR)、菌裂解直接PCR(Bacterium Direct-PCR)、免疫捕捉PCR(IC-PCR)、生物PCR(Bio-PCR)、SYBR Green染料实时荧光PCR(SYBR Green...采用传统生物学方法、显色培养基方法、自制三抗体夹心ELISA试剂盒(TAS-ELISA)、基因组直接PCR(DNA Direct-PCR)、菌裂解直接PCR(Bacterium Direct-PCR)、免疫捕捉PCR(IC-PCR)、生物PCR(Bio-PCR)、SYBR Green染料实时荧光PCR(SYBR Green Real time-PCR)、探针实时荧光PCR(TaqMan Real time-PCR)检测纯菌液及模拟带菌食品中的单核细胞增生性李斯特菌(Listeria monocytogenes,LM)。结果表明:传统生物学培养方法出现假阳性;TAS-ELISA、DNADirect-PCR、Bacterium Direct-PCR、IC-PCR最低检测限分别为106、102、105、104CFU/ml;显色培养基、SYBR Green Real time-PCR灵敏度达102CFU/ml;Bio-PCR、TaqMan Real time-PCR检测灵敏度最高,均为101CFU/ml。显色培养基、TAS-ELISA操作简单,适合大量样品的初检;IC-PCR具有灵敏、特异、快速、经济的优点,特别适合大体积样品中微量病原的检测;Bio-PCR、TaqMan Real time-PCR灵敏度高、特异性好,适合阳性样品的复检以及科研使用。展开更多
【研究目的】黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)的快速鉴定检测是控制该潜在危险性有害生物蔓延的有效途径。【方法】通过汁液摩擦接种、透射电镜观察、双抗夹心酶联免疫吸附测定法(DAS-ELISA)和RT-PCR方...【研究目的】黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)的快速鉴定检测是控制该潜在危险性有害生物蔓延的有效途径。【方法】通过汁液摩擦接种、透射电镜观察、双抗夹心酶联免疫吸附测定法(DAS-ELISA)和RT-PCR方法对该病毒进行鉴定;同时对RT-PCR反应体系及扩增程序进行优化,建立CGMMV免疫捕获RT-PCR(IC-RT-PCR)检测方法。【结果】血清学、生物学、电镜观察和分子生物学方法证明供试样本为黄瓜绿斑驳花叶病毒引致。IC-RT-PCR方法可以简化RNA的提取,降低对试验材料要求。【结论】IC-RT-PCR的建立为CGMMV的检测提供了操作更为简便、特异性更强的快速、准确的检测方法。展开更多
Maize chlorotic mottle virus (MCMV) infects maize plants and causes significant losses in corn production worldwide. In this study, purified MCMV particles were used as the immunogen to produce monoclonal antibodies (...Maize chlorotic mottle virus (MCMV) infects maize plants and causes significant losses in corn production worldwide. In this study, purified MCMV particles were used as the immunogen to produce monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs). Four murine MAbs (4B8, 8C11, 6F4, and 9G1) against MCMV were obtained through the hybridoma technology. The triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-immunobinding assay (DIBA), and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) using the MAb 4B8 were then developed for sensitive, specific, and rapid detection of MCMV in fields. MCMV could be detected in infected leaf crude extracts at dilutions of 1:327 680, 1:64000, and 1:3276800 (w/v, g/ml) by TAS-ELISA, DIBA, and IC-RT-PCR, respectively. One hundred and sixty-one maize field samples showing virus-like symptoms and sixty-nine symptomless maize field samples from ten different provinces of China were collected and screened for the presence of MCMV using the established serological methods. A phylogenetic tree was constructed based on the full length CP genes and Chinese MCMV isolates formed one branch with Thailand isolates. The detection results demonstrated that MCMV is one of most prevalent viruses infecting maize in the Yunnan and Sichuan provinces of China.展开更多
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 analy展开更多
文摘By Using the modified method to extract Rice dwarf virus virions from infected rice leaves,a concentration 3.854mg/mL of purified virus virions reached,and the Rabbits was immunized with purified virions,yielding high titer antiserum;1:20480 by the indirect enzyme-linked immunosorbent assay(I-ELISA)test.The antiserum successfully detected three rice samples from Kunming,Yuxi and Luliang by I-ELISA.Immunocapture polymerase chain reaction(IC-PCR),respectively was the utilized to show that serologic and molecular methods were successfully used for detection of Rice dwarf virus.
基金The Open Fundation Item of Agricultural Biotechnological Key Laboratory of Yunnan Province (2001B- 3)
文摘Two kinds of methods RT-PCR and IC-PCR were used to detect pathogen of virus disease of pumpkin and the sensitivity of the two methods was compared. The results showed that PRSV-W and CMV were detected in diseased samples gathered in Yunnan Province, while WMV and CMV were detected in diseased samples gathered in Heilongjiang Province. The sensitivity of RT-PCR is higher than that of IC-PCR, but the effect of IC-PCR in the specialization of bonding reaction and requisition for experiment material is better than that of RT-PCR.
文摘采用传统生物学方法、显色培养基方法、自制三抗体夹心ELISA试剂盒(TAS-ELISA)、基因组直接PCR(DNA Direct-PCR)、菌裂解直接PCR(Bacterium Direct-PCR)、免疫捕捉PCR(IC-PCR)、生物PCR(Bio-PCR)、SYBR Green染料实时荧光PCR(SYBR Green Real time-PCR)、探针实时荧光PCR(TaqMan Real time-PCR)检测纯菌液及模拟带菌食品中的单核细胞增生性李斯特菌(Listeria monocytogenes,LM)。结果表明:传统生物学培养方法出现假阳性;TAS-ELISA、DNADirect-PCR、Bacterium Direct-PCR、IC-PCR最低检测限分别为106、102、105、104CFU/ml;显色培养基、SYBR Green Real time-PCR灵敏度达102CFU/ml;Bio-PCR、TaqMan Real time-PCR检测灵敏度最高,均为101CFU/ml。显色培养基、TAS-ELISA操作简单,适合大量样品的初检;IC-PCR具有灵敏、特异、快速、经济的优点,特别适合大体积样品中微量病原的检测;Bio-PCR、TaqMan Real time-PCR灵敏度高、特异性好,适合阳性样品的复检以及科研使用。
文摘【研究目的】黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)的快速鉴定检测是控制该潜在危险性有害生物蔓延的有效途径。【方法】通过汁液摩擦接种、透射电镜观察、双抗夹心酶联免疫吸附测定法(DAS-ELISA)和RT-PCR方法对该病毒进行鉴定;同时对RT-PCR反应体系及扩增程序进行优化,建立CGMMV免疫捕获RT-PCR(IC-RT-PCR)检测方法。【结果】血清学、生物学、电镜观察和分子生物学方法证明供试样本为黄瓜绿斑驳花叶病毒引致。IC-RT-PCR方法可以简化RNA的提取,降低对试验材料要求。【结论】IC-RT-PCR的建立为CGMMV的检测提供了操作更为简便、特异性更强的快速、准确的检测方法。
基金supported by the National Natural Science Foundation of China (No. 31272015)the Ministry of Education of China (No. 313052)the Zhejiang Provincial Natural Science Foundation of China (No. Z3090039)
文摘Maize chlorotic mottle virus (MCMV) infects maize plants and causes significant losses in corn production worldwide. In this study, purified MCMV particles were used as the immunogen to produce monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs). Four murine MAbs (4B8, 8C11, 6F4, and 9G1) against MCMV were obtained through the hybridoma technology. The triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-immunobinding assay (DIBA), and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) using the MAb 4B8 were then developed for sensitive, specific, and rapid detection of MCMV in fields. MCMV could be detected in infected leaf crude extracts at dilutions of 1:327 680, 1:64000, and 1:3276800 (w/v, g/ml) by TAS-ELISA, DIBA, and IC-RT-PCR, respectively. One hundred and sixty-one maize field samples showing virus-like symptoms and sixty-nine symptomless maize field samples from ten different provinces of China were collected and screened for the presence of MCMV using the established serological methods. A phylogenetic tree was constructed based on the full length CP genes and Chinese MCMV isolates formed one branch with Thailand isolates. The detection results demonstrated that MCMV is one of most prevalent viruses infecting maize in the Yunnan and Sichuan provinces of China.
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 analy
