GFLV和GLRaV-3的发生及检测方法的研究
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摘要
以中国农业科学院郑州果树研究所国家葡萄资源圃的葡萄品种为试材，对葡萄扇叶病毒(GFLV)和葡萄卷叶相关病毒Ⅲ(GLRaV-3)进行了酶联免疫吸附测定(ELISA)的检测技术的研究。从阴、阳性对照及抗血清或抗体浓度的选择，到不同时期、检测部位对结果的影响，建立了一套完整的ELISA检测GFLV和GLRaV-3程序；本研究还对双抗体夹心酶联免疫吸附测定法(DAS-ELISA)和A蛋白夹心酶联免疫吸附测定法(PAS-ELISA)两种不同的检测方法进行了比较试验。发现，虽然DAS-ELISA在操作上较PAS-ELISA更为简便，但就结果而言，PAS-ELISA比DAS-ELISA的背景值或OD值都高，更易进行结果的判断。
结合ELISA和反转录聚合酶链式反应(RT-PCR)的特点，本研究引入了另外一种方法，即免疫捕捉反转录聚合酶链式反应(IC-RT-PCR)对GLRaV-3进行了检测。从免疫捕捉病毒、释放RNA到反转录成cDNA，最后扩增出了约300bp的预期目的片段。为了进一步明确PCR扩增的特异性，将PCR产物连接到pGEM-T载体上，转化大肠杆菌E.coli JM109菌株，通过蓝白斑筛选，获得重组质粒，提取质粒DNA，经酶切和PCR对重组质粒进行鉴定，结果证明得到了含有目的片段的重组质粒。经测序分析表明，其同源率达96.7％。由此证明IC-RT-PCR检测GLRaV-3结果准确可靠。最重要的是它弥补了ELISA易出现假阴性、假阳性以及RT-PCR提取RNA难等的缺点，不失为病毒检测的一种新方法。
在生长季节分三次对部分葡萄品种枝条上部、中部和下部叶片及韧皮部，进行双抗体夹心法(DAS-ELISA)、反转录聚合酶链式反应(RT-PCR)和免疫捕捉反转录聚合酶链式反应(IC-RT-PCR)三种方法检测卷叶病毒Ⅲ(GLRaV-3)的比较研究。结果表明：对于染有病毒并出现症状的品种，RT-PCR与IC-RT-PCR无论在任何时期、任何部位都检测出了GLRaV-3，但用DAS-ELISA对中、上部叶片的检出率分别为40％和60％；对无症状品种，DAS-ELISA的检出率明显低于RT-PCR和IC-RT-PCR方法。但对韧皮部的检测结果表明，三个时期采用任何方法均可以检测到该病毒。RT-PCR可以检测出RNA提取液为1：10~(-4)的GLRaV-3病毒，但它受到了不易提取RNA以及其它物质(如蛋白质，DNA等)干扰的影响，检测难度增加。从检测的可靠性来看，在春季所检测的8个无症品种中，RT-PCR和IC-RT-PCR全部检测出
了GLRaV一3，而DAS一ELISA仅检测出其中的6个。IC一RT一PCRt匕其它两种方法较
快，整个过程仅需8个小时。
The techniques of detecting grapevine fanleaf virus(GFLV) and grapevine leafroll-associated virus III(GLRa-3) by ELISA were studied. From the selection of negative, positive control and antibody or antiserum dilution times, to the result of detecting in different stage and site, We found an integrated detecting program on detecting GLRaV-3 and GFLV. In addition, we compared DAS-ELISA and PAS-ELISA in detecting GFLV. We found, though DAS-ELISA was simpler than PAS-ELISA in steps, PAS-ELISA is clearer than DAS-ELISA in conclusion.
We detected GLRaV-3 by immune capture reverese transcription ploymerase chain (IC-RT-PCR), form capture virus, release RNA, transcripe cDNA to the fragment of about 300bp PCR productions were cloned. To check the specificity and efficiency of GLRaV-3 cDNA fragment amplified, the fragment were isolated from agarose gels and ligated to pGEM-T, transfect E. coli JM109, the β -galactosides and ampicillin-resistant recombinant plasmids were obtained, these clones were further characterized by restriction enzyme digestion and PCR for the presence of the insert PCR products. The recombinant plasmids were then sequenced by the dideoxy chain termination methods. The results showed that there is 96.7% between what we obtained by IC-RT-PCR and the published. So we considered that the technique of IC-RT-PCR is exact and reliable and quarantine on grapevine in the future.
We compared the effectiveness of double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), reverse transcriptase polymerase chain reaction (RT-PCR) and immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) in detecting grapevine leafroll associated virus-3 (GLRaV-3) in infected symptomatic and nonsymptomatic grapevines throughout the growing season. Tissue
samples are from apical, middle and basal leaves. We found: DAS-ELISA, IC-RT-PCR and RT-PCR detected GLRaV-3 in nearly all samples of basal leaves collected throughout the growing season. In contrast, detection of GLRaV-3 in nonsymptomatic vines was erratic regardless of the detection method that was used, and the results were affected on season and tissue samples. Based on the results, we recommend that the phloem of canes from vines or from dormant cutting be used. Moreover, we thought RT-PCR could detect the fewer viruses than DAS-ELISA and IC-RT-PCR, but it was difficult to extract the RNA from the grapevine tissue, and the result was affected by protein, DNA etc from the plant tissue. IC-RT-PCR and RT-PCR were more sensitive than DAS-ELISA. In additional, IC-RT-PCR was faster and more effective detecting GLRaV-3.
结合ELISA和反转录聚合酶链式反应(RT-PCR)的特点，本研究引入了另外一种方法，即免疫捕捉反转录聚合酶链式反应(IC-RT-PCR)对GLRaV-3进行了检测。从免疫捕捉病毒、释放RNA到反转录成cDNA，最后扩增出了约300bp的预期目的片段。为了进一步明确PCR扩增的特异性，将PCR产物连接到pGEM-T载体上，转化大肠杆菌E.coli JM109菌株，通过蓝白斑筛选，获得重组质粒，提取质粒DNA，经酶切和PCR对重组质粒进行鉴定，结果证明得到了含有目的片段的重组质粒。经测序分析表明，其同源率达96.7％。由此证明IC-RT-PCR检测GLRaV-3结果准确可靠。最重要的是它弥补了ELISA易出现假阴性、假阳性以及RT-PCR提取RNA难等的缺点，不失为病毒检测的一种新方法。
在生长季节分三次对部分葡萄品种枝条上部、中部和下部叶片及韧皮部，进行双抗体夹心法(DAS-ELISA)、反转录聚合酶链式反应(RT-PCR)和免疫捕捉反转录聚合酶链式反应(IC-RT-PCR)三种方法检测卷叶病毒Ⅲ(GLRaV-3)的比较研究。结果表明：对于染有病毒并出现症状的品种，RT-PCR与IC-RT-PCR无论在任何时期、任何部位都检测出了GLRaV-3，但用DAS-ELISA对中、上部叶片的检出率分别为40％和60％；对无症状品种，DAS-ELISA的检出率明显低于RT-PCR和IC-RT-PCR方法。但对韧皮部的检测结果表明，三个时期采用任何方法均可以检测到该病毒。RT-PCR可以检测出RNA提取液为1：10~(-4)的GLRaV-3病毒，但它受到了不易提取RNA以及其它物质(如蛋白质，DNA等)干扰的影响，检测难度增加。从检测的可靠性来看，在春季所检测的8个无症品种中，RT-PCR和IC-RT-PCR全部检测出
了GLRaV一3，而DAS一ELISA仅检测出其中的6个。IC一RT一PCRt匕其它两种方法较
快，整个过程仅需8个小时。
The techniques of detecting grapevine fanleaf virus(GFLV) and grapevine leafroll-associated virus III(GLRa-3) by ELISA were studied. From the selection of negative, positive control and antibody or antiserum dilution times, to the result of detecting in different stage and site, We found an integrated detecting program on detecting GLRaV-3 and GFLV. In addition, we compared DAS-ELISA and PAS-ELISA in detecting GFLV. We found, though DAS-ELISA was simpler than PAS-ELISA in steps, PAS-ELISA is clearer than DAS-ELISA in conclusion.
We detected GLRaV-3 by immune capture reverese transcription ploymerase chain (IC-RT-PCR), form capture virus, release RNA, transcripe cDNA to the fragment of about 300bp PCR productions were cloned. To check the specificity and efficiency of GLRaV-3 cDNA fragment amplified, the fragment were isolated from agarose gels and ligated to pGEM-T, transfect E. coli JM109, the β -galactosides and ampicillin-resistant recombinant plasmids were obtained, these clones were further characterized by restriction enzyme digestion and PCR for the presence of the insert PCR products. The recombinant plasmids were then sequenced by the dideoxy chain termination methods. The results showed that there is 96.7% between what we obtained by IC-RT-PCR and the published. So we considered that the technique of IC-RT-PCR is exact and reliable and quarantine on grapevine in the future.
We compared the effectiveness of double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), reverse transcriptase polymerase chain reaction (RT-PCR) and immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) in detecting grapevine leafroll associated virus-3 (GLRaV-3) in infected symptomatic and nonsymptomatic grapevines throughout the growing season. Tissue
samples are from apical, middle and basal leaves. We found: DAS-ELISA, IC-RT-PCR and RT-PCR detected GLRaV-3 in nearly all samples of basal leaves collected throughout the growing season. In contrast, detection of GLRaV-3 in nonsymptomatic vines was erratic regardless of the detection method that was used, and the results were affected on season and tissue samples. Based on the results, we recommend that the phloem of canes from vines or from dormant cutting be used. Moreover, we thought RT-PCR could detect the fewer viruses than DAS-ELISA and IC-RT-PCR, but it was difficult to extract the RNA from the grapevine tissue, and the result was affected by protein, DNA etc from the plant tissue. IC-RT-PCR and RT-PCR were more sensitive than DAS-ELISA. In additional, IC-RT-PCR was faster and more effective detecting GLRaV-3.
引文
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