甘藷羽狀斑駁病毒反轉錄恆溫環狀擴增檢測技術 (RT-LAMP) 之建立與應用
Other Title
Establishment and Application of Reverse Transcription Loop-Mediated Isothermal Amplification Assay (RT-LAMP) for the Detection of Sweet Potato Feathery Mottle Virus in Sweet Potato
Journal
台灣農業研究
Journal Volume
73
Journal Issue
2
Pages
113-122
Date Issued
2024-06-04
Author(s)
Abstract
病毒病害為限制甘藷生產的重要因子，其中由馬鈴薯Y 病毒屬 (Potyvirus) 中之甘藷羽狀斑駁病毒 (sweet potato feathery mottle virus; SPFMV) 引起之病害，為全球甘藷栽培區廣泛發生之病毒病害。甘藷健康種苗的生產與早期的病毒診斷鑑定皆需仰賴快速可靠的病毒檢測技術，亦是有效防治病毒病害的重要關鍵。本研究建立單步驟反轉錄恆溫環狀擴增 (reverse transcription loop-mediated isothermal amplification; RT-LAMP) 檢測技術，藉以檢測SPFMV，並利用比色法，藉hydroxy naphthol blue 作為指示染劑，以利肉眼辦別反應之結果。以此技術實際檢測罹病甘藷植物總量RNA，最佳反應條件為63℃、45–60 min；可檢測的最低RNA 濃度為237.9 × 10-5 ng μL-1，相較於反轉錄聚合酵素連鎖反應 (reverse transcription polymerase chain reaction; RT-PCR)可檢測的最低RNA 濃度為237.9 × 10-3 ng μL-1，顯示RT-LAMP 之檢測靈敏度較RT-PCR 高100 倍。此外，該RT-LAMP 技術亦成功應用於田間甘藷之SPFMV 檢測，顯示RT-LAMP 檢測技術是具有快速、專一且靈敏之SPFMV 檢測方法，未來可優化目前種苗檢定技術與提供給甘藷業者使用。
Viral diseases are a major constraint to sweet potato production. Sweet potato feathery mottle virus (SPFMV), a member of the genus Potyvirus, is the most widespread virus infecting sweet potato plants worldwide. The production of healthy sweet potato seedlings and the diagnosis of plant viruses at early stage rely on rapid and reliable viral detection technology, which is critical for effective viral prevention and control. In this study, a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting SPFMV. The RT-LAMP assay has been optimized to use hydroxy naphthol blue (HNB) for visual colorimetric distinction of positive and negative samples. Using total plant RNA of sweet potato as the template, the optimal reaction conditions were at 63℃ for 45–60 min. The detection limit of the RT-LAMP assay for SPFMV was 237.9 × 10-5 ng μL-1, while that of reverse transcription polymerase chain reaction (RT-PCR) was 237.9 × 10-3 ng μL-1, indicating that the RT-LAMP assay developed was 100 times more sensitive than RT-PCR. The RTLAMP assay was also successfully applied to the detection of SPFMV in field samples. In conclusion, the RT-LAMP assay developed in this study was a rapid, specific and sensitive method for the detection of SPFMV in field samples of sweet potato.
Subjects
甘藷羽狀斑駁病毒
反轉錄恆溫環狀擴增檢測技術
甘藷
Sweet potato feathery mottle virus
Reverse-transcription loop-mediated isothermal amplification
sweet potato
Publisher
農業試驗所
Type
journal article
File(s)![Thumbnail Image]()
Loading...
Name
73-2-4.pdf
Size
1.77 MB
Format
Adobe PDF
Checksum
(MD5):6e827a020a46fa8e12873977a918cdc8