應用核酸探針系統檢測瓜類褪綠黃化病毒

A DNA Probing System for the Detection of ＂Cucurbit Chlorotic Yellows Virus＂

關政平(Cheng-Ping Kuan) ； 曾清山(Ching-Shan Tseng) ； 張文學(Wen-Shiue Chang) ； 石信德(Hsin-Der Shih) ； 鄭櫻慧(Ying-Huey Cheng) ； 鄧汀欽(Ting-Ching Deng)

《台灣農業研究》 63卷4期 (2014/12) Pp. 259-266

https://doi.org/10.6156/JTAR/2014.06304.01

摘要

本研究以微珠核酸探針系統應用於瓜類褪綠黃化病毒(CCYV)之檢測，並進行田間洋香瓜是否感染CCYV之測試。此微珠核酸探針系統之檢測包含可擴增CCYV病毒鞘蛋白(CP)基因及一組植物葉綠體來源基因(ndhB)，分別設計一組引子對，進行反轉錄聚合酵素連鎖反應及微珠核酸探針系統反應。微珠核酸探針系統設計上，尚包含專一性探針分別與基因擴增內部之序列核酸雜配結合之序列。利用核酸探針結合螢光染劑(R-PE)由冷光檢測分析系統(LiquiChip)分析螢光訊號作判讀，結果顯示具有高度專一性靈敏度，較使用傳統RT-PCR的靈敏度提高約100倍。此法預期將有助於檢測CCYV感染的洋香瓜種苗驗證。

關鍵字

瓜類褪綠黃化病毒(CCYV) ； 核酸探針 ； 檢測

並列摘要

In this paper, a PCR assay coupled with microspheres to rapidly screen field samples for ＂Cucurbit chlorotic yellows virus＂ (CCYV) survey on melons was developed. The RT-PCR was designed to have the same binding sites for the forward and reverse primers of the CCYV viral coat protein (CP) gene as the target amplicon, but it had a unique internal sequence used for the probe site. The amplification of the CCYV viral RNA and the detection by LiquiChip were monitored with two different fluorescent probes in a multiplex format, one specific for the CCYV CP gene and the other for the plant chloroplast source gene, ndhB. The detection sensitivity of multiplex RT-PCR by LiquiChip was 100 times higher than only detection by RT-PCR for CCYV. The hybridized beads were processed and analyzed on LiquidChip instrument, which detected the presence of CCYV RNA and quantity of each PCR product from melon samples. Therefore, it may have great advantage to the certification program of melons.

並列關鍵字

＂Cucurbit chlorotic yellows virus＂ (CCYV) ； Nucleic acid probe ； Detection