Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus.
Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus.
复制标题
基于单克隆抗体的黄瓜绿斑驳花叶病毒血清学检测方法
DOI：
10.1186/1743-422x-8-228
复制
发表时间：
2011-05-15
期刊：
影响因子：
4.8
通讯作者：
Wu J
中科院分区：
文献类型：
--
作者：
Shang H;Xie Y;Zhou X;Qian Y;Wu J
黄瓜绿色斑驳花叶病毒（Cucumber green mottle mosaic virus，CGMMV）是烟草花叶病毒属（Tobamovirus）的成员，可通过种子传播，侵染多种瓜类植物，造成黄瓜和西瓜的严重减产。本文介绍了抗原包被平板酶联免疫吸附试验等五种血清学方法（ACP-ELISA），三抗体夹心酶联免疫吸附试验TAS-ELISA、斑点免疫结合试验（DBIA）、直接组织印迹免疫试验（DTBIA）和免疫捕获逆转录聚合酶链反应（RT-PCR）结果以纯化的CGMMV颗粒为免疫原，制备了6株鼠源性单克隆抗体。五种血清学方法建立了使用单克隆抗体4 H1和检测灵敏度进行了比较，使用纯化的制剂和感染的植物组织提取物。ACP-ELISA的检测灵敏度为0.16 ng，而TAS-ELISA的最低检测灵敏度为0.04 ng，高于ACP-ELISA。TAS-ELISA和DBIA检测CGMMV的灵敏度相近，比ACP-ELISA高4倍。IC-RT-PCR检测病毒的灵敏度最高，可检测到0.1pg的纯化病毒。结论所建立的ACP-ELISA、TAS-ELISA、DBIA和DTBIA检测方法均适用于CGMMV的大规模现场检测，而IC-RT-PCR检测方法更敏感，更适合于CGMMV基因组信息的获取。
BackgroundCucumber green mottle mosaic virus (CGMMV), a member of the genusTobamovirus, can be transmitted by seeds and infects many cucurbit species, causing serious yield losses in cucumber and watermelon plants. In this paper, five serological methods including antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA), triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), Dot-immunobinding assay (DBIA), direct tissue blot immunoassay (DTBIA) and immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) were described for detection and diagnosis of CGMMV.ResultsUsing the purified CGMMV particles as immunogens, six murine monoclonal antibodies (MAbs) were produced. Five serological methods were established using the MAb 4H1 and detection sensitivity was compared using purified preparations and infected-plant tissue extracts. The detection sensitivity of ACP-ELISA was 0.16 ng of purified CGMMV, whereas TAS-ELISA was more sensitive than ACP-ELISA with a minimum detection of 0.04 ng of purified CGMMV. The sensitivities of TAS-ELISA and DBIA were similar for detecting CGMMV in infected-plant tissue extracts, and were four times higher than ACP-ELISA. The IC-RT-PCR was the most sensitive method, which could detect as little as 0.1 pg of purified virus. The detection sensitivity of IC-RT-PCR for CGMMV-infected plant tissues was about 400 times higher than that of TAS-ELISA and DBIA.ConclusionsThe established ACP-ELISA, TAS-ELISA, DBIA and DTBIA are suitable for routine CGMMV detection of large-scale samples in the field survey, while IC-RT-PCR is more sensitive and suitable for acquiring information about the viral genome.