高灵敏度血清学方法检测番茄植株和烟粉虱中的番茄黄曲叶病毒。

Highly sensitive serological methods for detecting tomato yellow leaf curl virus in tomato plants and whiteflies.

机构信息

State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang 310058, China.

出版信息

Virol J. 2013 May 6;10:142. doi: 10.1186/1743-422X-10-142.

DOI:10.1186/1743-422X-10-142

PMID:23647724

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3684515/

Abstract

BACKGROUND

Tomato yellow leaf curl virus (TYLCV) is a member of the genus Begomovirus in the family Geminiviridae, which causes severe losses in tomato production in tropic and subtropic regions.

METHODS

The purified TYLCV virions were used as the immunogen to produce monoclonal antibodies (MAbs) using the hybridoma technology. MAb-based dot enzyme-linked immunosorbent assay (dot-ELISA) and direct tissue blot immunoassay (DTBIA) were developed for sensitive, simple, and rapid detection of TYLCV in field tomato and whitefly (Bemisia tabaci) samples collected from TYLCV prevalent provinces in China.

RESULTS

Using the hybridoma technology, six murine MAbs (1C4, 8D10, 6E3, 2F2, 3F4 and 4G3) against TYLCV were prepared. Using the MAb 1C4, dot-ELISA and DTBIA were then established for detecting TYLCV in field tomato and whitefly samples collected from TYLCV prevalent provinces in China. The dot-ELISA could detect TYLCV in infected tissue crude extract diluted at 1:5,120 (w/v, g mL-1), and in viruliferous whitefly homogenate diluted at 1:128 (individual whitefly/μL), respectively. Field tomato samples (n=487) and whitefly samples (n=110) from TYLCV prevalent districts in China were screened for the presence of TYLCV using the two developed methods, and the results were further confirmed by PCR and nucleotide sequencing. The survey revealed that TYLCV is widespread on tomato plants in Zhejiang, Shandong and Henan provinces in China.

CONCLUSIONS

The developed dot-ELISA is very suitable for the routine detection of TYLCV in field tomato and whitefly samples, and the DTBIA is more suitable for the routine detection of TYLCV in large-scale tomato plant samples collected from TYLCV prevalent areas.

摘要

背景

番茄黄曲叶病毒（TYLCV）是双生病毒科的菜豆金色花叶病毒属的一员，在热带和亚热带地区会对番茄生产造成严重损失。

方法

纯化的 TYLCV 病毒粒子被用作免疫原，通过杂交瘤技术产生单克隆抗体（MAbs）。基于 MAb 的点酶联免疫吸附测定（dot-ELISA）和直接组织印迹免疫分析（DTBIA）被开发用于敏感、简单和快速检测中国 TYLCV 流行省份田间番茄和烟粉虱（Bemisia tabaci）样品中的 TYLCV。

结果

使用杂交瘤技术，制备了针对 TYLCV 的 6 株鼠源 MAbs（1C4、8D10、6E3、2F2、3F4 和 4G3）。使用 MAb 1C4，建立了 dot-ELISA 和 DTBIA 来检测中国 TYLCV 流行省份田间番茄和烟粉虱样品中的 TYLCV。dot-ELISA 可检测感染组织粗提物稀释 1:5,120（w/v，g mL-1）和带毒烟粉虱匀浆稀释 1:128（单个烟粉虱/μL）的 TYLCV。使用两种方法对来自中国 TYLCV 流行地区的 487 份田间番茄样品和 110 份烟粉虱样品进行 TYLCV 存在情况筛查，结果通过 PCR 和核苷酸测序进一步确认。调查显示，TYLCV 在浙江、山东和河南等省的番茄植株中广泛存在。