鑑定邊境攔截之陸蓮花微嵌紋病毒（Ranunculus mild mosaic virus）及其檢測試劑之製備與應用

Identification of Ranunculus Mild Mosaic Virus on Ranunculus asiaticus Collected from Border Intercepted Ranunculus and the Development and Application of the Virus Detection Reagents

陳金枝(Chin-Chih Chen) ； 廖家翌(Jia-Yi Liao) ； 廖敏伶(Ming-Ling Liao) ； 劉逸琪(Yu-Chi Liu) ； 江芬蘭(Fen-Lang Chiang)

《台灣農業研究》 71卷1期 (2022/03) Pp. 73-86

https://doi.org/10.6156/JTAR.202203_71(1).0006

摘要

本研究鑑定進口陸蓮花（Ranunculus asiaticus）切花上引起葉片嵌紋徵狀之病毒，病葉樣品為行政院農業委員會動植物防疫檢疫局新竹分局於輸入檢疫取樣之荷蘭進口陸蓮花。初步以花卉病毒多元抗體進行間接式酵素連結免疫吸附反應（indirect enzyme-linked immunosorbent assay; indirect ELISA）檢測，受測樣品均與potyvirus單元抗體（Agdia Inc., Elkhart, IN, USA）產生正反應，並能與potyvirus簡併式引子對[HRP-5/Oligo-dT_((14))]於RT-PCR反應中產生預估大小約1.3 kbp之核酸片段，經選殖與定序鑑定後，乃屬於ranunculus mild mosaicvirus（RanMMV）之核酸序列，並與GenBank上已登錄之RanMMV（Accession No. LC387972）之鞘蛋白核苷酸及胺基酸序列有高達98.6%以上之相同度，屬相同病毒之分離株。於reverse transcription-polymerase chain reaction（RT-PCR）檢測中，本研究所設計的RanMMV引子對（RanMM-u/RanMM-d）均可由葉片呈現嵌紋徵狀的陸蓮花切花和罹病種球組織檢出1.04 kbp之核酸片段產物。進一步將解得之RanMMV鞘蛋白核酸序列共825個核苷酸以基因合成法構築於pET28a（＋）表現載體上，再於Escherichia coli BL21宿主中進行鞘蛋白之誘導（分子量約30.5 kDa），並將其作為抗原以製備RanMMV之多元抗體，可成功用於進口陸蓮花切花及種球上對RanMMV之檢測。本研究為首次於邊境截獲國外的陸蓮花病毒RanMMV，國內尚未有此病毒之發生，本研究成功研製之RanMMV核酸及免疫檢測技術，將可提升對進口陸蓮花之邊境檢疫效能。

關鍵字

陸蓮花 ； 馬鈴薯Y屬病毒 ； 多元抗體 ； RT-PCR檢測

並列摘要

Ranunculus asiaticus cut flowers with mosaic symptoms imported from The Netherlands were intercepted by quarantine at the border customs. The potential causal agents were identified and the detection tools were developed in this study. Indirect enzyme-linked immunosorbent assay (indirect ELISA) was performed with various antibodies against different ornamental viruses preserved in the laboratory. All the tested samples were positively reacted with the commercial potyvirus monoclonal antibody (produced by Agdia Inc., Elkhart, IN, USA) and could be positively detected with the potyvirus-degenerated primers (HRP-5/Oligo-dT_((14)) to amplify a 1.3-kbp amplicon in reverse transcription- polymerase chain reactions (RT-PCRs). Sequencing data showed the nucleic acid sequences of the amplified amplicons were similar to that of ranunculus mild mosaic virus (RanMMV). Both of the nucleotide and deduced amino acid sequences of the cloned cDNA shared more than 98.6% identities with that of RanMMV coat protein gene (GenBank Accession No. LC387972) indicating the intercepted virus is a RanMMV isolate. The further designed RanMMV primer pair (RanMM-u/ RanMM-d) can effectively detect the RanMMV on imported mosaic cut flowers and bulb tissues of R. asiaticus, and generate a predicted 1.04 kbp amplicon. The cDNA of the RanMMV coat protein open reading frame (CP ORF), containing 825 nucleotides, was constructed in the expression vector of pET28a(+) by artificial gene synthesis, and then the fusion protein (approximately Mt. 30.5 kDa) was expressed in the Escherichia coli BL21 host. The resulting RanMMV coat protein was used as an antigen to prepare a polyclonal antibody against RanMMV. The produced polyclonal antibody against RanMMV can successfully detect RanMMV on the imported R. asiaticus in immunoassays. RanMMV has not been found in Taiwan and this is the first interception case of the virus at the border. The detection reagents of RanMMV have been successfully developed in this study which might be helpful in improving the efficiency of border quarantine and inspection on imported Ranunculus materials.

並列關鍵字

Ranunculus asiaticus ； Potyvirus ； Polyclonal antibody ； RT-PCR detection