Development of serological procedures for sensitive, rapid detection of Cucumber mosaic virus in Lilium

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• 作者：Han Sol Kim ; Dong Wan Kim ; Yong-Tae Jung
• 关键词：Additional key wordscoat protein ; CMV ; IC ; RT ; PCR ; ID ; ELISA ; polyclonal antisera
• 刊名：Horticulture, Environment, and Biotechnology
• 出版年：2016
• 出版时间：December 2016
• 年：2016
• 卷：57
• 期：6
• 页码：633-639
• 全文大小：
• 刊物主题：Life Sciences, general; Plant Breeding/Biotechnology; Plant Physiology; Agriculture; Plant Ecology;
• 出版者：Korean Society for Horticultural Science
• ISSN：2211-3460
• 卷排序：57

文摘

Cucumber mosaic virus (CMV) causes severe agricultural losses in Korea and has a very large host range. To produce virus-free plants, it is necessary to develop efficient, sensitive virus detection methods. In this study, we collected leaf samples from Lilium showing characteristic symptoms of CMV infection from Gang-won Province, Korea in 2011 and amplified the coat protein (CP) gene from CMV from the samples by RT-PCR. Three CMV isolates were found to belong to subgroup IA based on the nucleotide sequence of the CP gene. We cloned the complete CP gene into the pET21d(+) expression vector to generate recombinant coat proteins. After expressing His-tagged coat proteins in Escherichia coli strain BL21 (DE3) by IPTG induction, we purified the proteins using Ni-NTA agarose beads and used the purified CMV recombinant CP for antiserum production. The resulting polyclonal antisera specifically recognized CMV from infected lily samples, as determined by indirect enzyme-linked immunosorbent assay (ID-ELISA) and Western blotting assays. In addition, we developed an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) assay using the polyclonal antiserum to enable sensitive, reliable, cost-effective detection of CMV. This study demonstrates that three CMV isolates belong to subgroup IA, and that CMV-specific PAbs can be used to detect CMV in epidemiological studies.