胡瓜嵌紋病毒火球花分離株之鑑定與檢測

Identification and Detection of ＂Cucumber mosaic virus＂ Isolated from Bloody Lily (＂Haemanthus multiflorus＂)

陳金枝(Chin-Chih Chen) ； 江芬蘭(Fen-Lang Chiang)

《台灣農業研究》 64卷1期 (2015/03) Pp. 74-79

https://doi.org/10.6156/JTAR/2015.06401.08

摘要

本研究由火球花（Haemanthus multiflorus Martyn.）葉片呈現黃斑病徵者取其罹病組織，經由汁液機械接種於奎藜（Chenopodium quinoa Willd.）葉片所得之單斑（single lesion）而獲得YS1分離株。續於間接式酵素連結免疫反應（Indirect enzyme-linked immunosorbent assay; indirect-ELISA）中，火球花自然罹病葉、YS1分離株所感染之奎藜局部病斑及菸草（Nicotiana benthaminana Domin）系統性嵌紋病葉等受測樣品，均可與胡瓜嵌紋病毒（Cucumber mosaic virus; CMV）多元抗體產生正反應。而於反轉錄－聚合酶鏈鎖反應（reverse transcriptionpolymerase chain reaction; RT-PCR）反應中，上述3種受測樣品之全量核酸，均可與CMV引子對（CMV-up/ CMV-dw）產生預期大小約485 bp之核酸片段。YS1之鞘蛋白（YS1-CP）核酸序列登錄於GenBank取得核酸分子序號為KM066122，YS1-CP之胺基酸序列與其他已知CMV之相同度均高於90%。由血清學及核酸分子特性之鑑定結果，顯示由火球花分離而得之YS1為CMV之分離株。CMV寄主範圍廣泛，本研究之結果乃CMV之新寄主紀錄。本研究分別以TPS（Thomson & Dietzgen 1995）及自製之PSE緩衝液快速萃取罹病植物組織之全量核酸，以其為模板進行RT-PCR，均可成功增幅出YS1病毒之預估核酸片段，結果證實此種核酸快速萃取法可用於CMV之核酸快速檢測，具有相當高之應用潛力。

關鍵字

胡瓜嵌紋病毒 ； 火球花 ； 免疫分析 ； RT-PCR快速檢測

並列摘要

A yellow spot symptom observed on leaves of blood lily (Haemanthus multiflorus Martyn.) plant was used for virus identification. Later, a virus isolate (YS1) was obtained from single-lesion isolation by mechanical inoculation of this diseased tissue extracts to leaves of Chenopodium quinoa Willd. Extracts of the diseased blood lily, local lesions on leaf of C. quinoa and systemic mosaic leaf tissues of Nicotiana benthamiana caused by YS-1 were all reacted strongly with the antiserum against Cucumber mosaic virus (CMV) in indirect enzyme-linked immunosorbent assay. An expected size of 485-bp DNA fragment was amplified from total RNAs of the field blood lily sample, YS1-infected C. quinoa and N. benthamiana by reverse transcription-polymerase chain reaction (RT-PCR) using the primer pair of CMV-up/CMV-dw. The nucleotide sequence of coat protein (CP) region of YS1 isolate was determined and submitted to the GenBank with the accession number KM066122. The amino acid sequence of YS1-CP shared higher than 90% identity with those of known CMV isolates. Based on the results of serological assay and molecular evidence, it indicated that YS1 isolated from blood lily was an isolate of CMV. Although CMV distributes worldwide and has a large host range. This is the first report to demonstrate that blood lily is a nature host of CMV in Taiwan. Total RNAs of YS1- infected plant tissues obtained by the rapid extraction way with TPS (Thomson & Dietzgen 1995) or PSE buffer (prepared in this study) were all successfully applied on the RT-PCR analysis of YS1. The potential use of the rapid extraction of plant total RNAs for RT-PCR amplification of CMV is proved in this study.

並列關鍵字

＂Cucumber mosaic virus＂ (CMV) ； Blood lily (＂Haemanthus multiflorus＂ Martyn.) ； Serological assay ； Rapid detection with RT-PCR