Cymbidium ringspot virus核酸及血清檢測法之建立及其應用

Development and Application of the Molecular and Serological Detection Methods for Cymbidium ringspot virus

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

《台灣農業研究》 66卷3期 (2017/09) Pp. 202-218

https://doi.org/10.6156/JTAR/2017.06603.04

摘要

Cymbidium ringspot virus (CymRSV) 為國外已有發生之病毒，目前國內尚未有此病毒之發生紀錄。本研究針對此病毒進行其核酸及血清檢測試劑製備，以應用於蘭花病毒監測。核酸檢測法方面，以對應CymRSV 鞘蛋白 (coat protein; CP) 之核苷酸序列設計2 組引子對，應用於反轉錄- 聚合酶鏈鎖反應法 (reversetranscription-polymerase chain reaction; RT-PCR)。CyRcpu/CyRcpd 引子對可增幅出涵蓋CymRSV-CP 基因全長及其兩端序列共約1370 bp 之片段，CyRu/CyRd 引子對可增幅出CymRSV-CP 基因高保留區約540 bp 之片段，於RT-PCR 中均可成功檢測到市售之CymRSV RNA 標準對照品 (DSMZ 出品)，獲得與預估分子量相符之核酸增幅產物。CyRu/CyRd 引子對於60℃煉合溫度下，於RT-PCR 法中可專一性檢出CymRSV。另外，針對CymRSV-CP 基因序列設計4 條引子 (CyR-FIP/CyR-BIP/CyR-F3 /CyR-B3)，以反轉錄- 恆溫環形核酸增幅法(reverse transcription Loop-mediated isothermal amplification; RT-LAMP)，可於65℃恆溫條件下於1 h 反應中成功擴增此病毒核酸分子，並直接以目視法判讀反應結果，可專一性檢出CymRSV 之標準對照品，且有高於RT-PCR 約25 倍之靈敏度。在免疫檢測法方面，以人工基因合成CymRSV 全長度鞘蛋白核酸片段，並構築於pET28b 表現載體，於Escherichia. coli (Rosetta) 細菌宿主中進行蛋白表現，獲得分子量約41.8 kDa 之融合性表現蛋白 (BEP189)，且可與2 種國外市售之CymRSV 多元抗體產生正反應；以BEP189 做為抗原所製備出之多元抗體 (CymRSV#189) 於間接式-酵素連結免疫吸附反應 (indirect enzyme-linked immunosorbent assay;indirect ELISA) 及西方墨點法 (western blotting) 反應中，均可與其同源之表現蛋白及國外市售之CymRSV 免疫檢測標準品產生正反應，但不與健康蘭花組織以及9 種其他蘭花病毒產生正反應，自製之多元抗體可成功應用於CymRSV 之專一性免疫檢測與鑑別。本研究首次開發RT-LAMP 法於CymRSV 之核酸快檢以輔助對此病毒之鑑定，並研發可實務應用於蘭花上對CymRSV 監測之RT-PCR 技術，且自製之多元抗體可有效地鑑別CymRSV。

關鍵字

CymRSV ； 血清檢測 ； RT-PCR檢測 ； 反轉錄-恆溫環形核酸增幅法

並列摘要

Cymbidium ringspot virus (CymRSV) is recorded in foreign countries, but not found in Taiwan. The molecular and serological detection reagents for the inspection of CymRSV on orchids were developed in this study. In molecular detection, two primer pairs for reverse transcription-polymerase chain reaction (RT-PCR) were designed based on the nucleotide sequences of CymRSV coat protein (CP). The primer pair CyRcpu/CyRcpd was designed for amplifying the full-length CP gene and its flanking borders. The primer pair CyRu/CyRd was used to amplify the conserved region of the CymRSV-CP gene. In RT-PCR, the expected 1370-bp and 540-bp DNA fragments were amplified by the primer pairs CyRcpu/CyRcpd and CyRu/CyRd, respectively, from the commercial DSMZ-CymRSV RNA. The annealing temperature under the 60℃ , the primer pair CyRu/CyRd was used to specifically detect CymRSV in RT-PCR. A set of four primers (CyR-FIP/CyR-BIP/CyR-F3/ CyR-B3) was successfully used to amplify CymRSV RNA under 65℃ for 1 h in reverse transcription loop-mediated isothermal amplification (RT-LAMP), and the results were able to be determined by naked eye observation. RT-LAMP with a specificity for CymRSV detection and has a 25-time higher sensitivity than RT-PCR assay. In serological detection, the predicted 41.8 kDa of the bacterial expressed fusion protein (BEP189) was positively reacted to two commercial CymRSV antibodies. The CymRSV antiserum (#189) was prepared from the rabbit immunized with the bacterial-expressed CymRSV-CP. In the detections of CymRSV by the ways of indirect enzyme-linked immunosorbent assay (indirect ELISA) and western blotting, the prepared antibody #189 specifically reacted with its expressed CP as well as with the commercial controls of CymRSV, but not reacted to the healthy orchid tissues and other 9 viruses on orchids. This is the first report to develop the RT-LAMP method for detecting CymRSV and to apply the RT-PCR assay for inspecting CymRSV on orchids. Moreover, the prepared polyclonal antibody against CymRSV is efficient to differentiate CymRSV from other 9 tested orchid viruses.

並列關鍵字

Cymbidium ringspot virus (CymRSV) ； Serological detection ； RT-PCR detection ； RTLAMP