Rapid Detection of Phytoplasma Associated with Paulownia Witches'-broom Using a Nonradioactive DNA Probe and a PCR-based Assay

應用核酸探針與PCR技術快速偵測泡桐簇葉病菌質體

吳孟玲(Meng-Ling Wu) ； 張東柱(Tun-Tschu Chang) ； 王涼綢(Laing-Chour Wang) ； 傅春旭(Chuen-Hsu Fu)

《台灣林業科學》 17卷2期 (2002/06) Pp. 123-133

https://doi.org/10.7075/TJFS.200206.0123

摘要

由非培養性植物菌質體(phytoplasma)引起之泡桐簇葉病(paulownia witches'-broom, PaWB)，由於蟲媒傳播及系統性感染，在臺灣普遍嚴重發生爲害。此病害在無性繁殖泡桐株上易傳播流行，爲害更爲嚴重，成爲生産之限制因子。以DNA選殖的方法所開發之非放射性DNA探針，具有高敏感度與高特異性，已應用於泡桐簇葉病茵質體(PaWBP)之偵測。經篩選所得之一特異性DNA片段，大小爲762bp，經由PCR法將生物素(biotin)標識於此片段而製成探針，並應用於PaWBP點漬雜配偵測法。同時再經由核酸序列定序，進一步選定PaWBP之專一性引子對，開發高敏感度、快速且正確的PCR偵測技術，所複製之PaWBP專一性PCR片段大小爲510bp；配合簡易的核酸抽取法，整套PCR偵測流程僅須六小時即可完成，此技術可爲泡桐簇葉病之診斷提供更快速有效的方法。

關鍵字

泡桐簇葉病 ； 植物菌質體 ； 非放射性核酸探針 ； 聚合酵素鏈鎖反應

並列摘要

Due to insect-borne transmission and systemic infection, paulownia witches'-broom (PaWB) caused by phytoplasma, has created a serious epidemic in Taiwan. The disease seriously affects paulownia plants, and has become a limiting factor for paulownia cultivation. A highly sensitive and specific nonradioactive DNA probe developed from DNA cloning methods has been used to detect PaWB phytoplasma (PaWBP) in infected paulownia hosts. One of the clones containing a 762-bp PaWBP-specific DNA fragment was labeled with biotinylated nucleotides by a PCR-labeling technique. For more rapid and sensitive detection, an assay based on the polymerase chain reaction (PCR) has been developed using primers derived from sequences of the cloned DNA fragment of PaWBP to detect PaWBP in paulownia. One set of the primer pairs (named the 510-primer pair), which generates a 510-bp PaWBP-specific fragment from total DNA templates purified from diseased paulownia plants, was tested and chosen for PCR amplification. A simple method for the extraction of DNA from paulownia tissues was also developed to prepare template DNA for PCR. This detection protocol, which can be completed within 6 h, offers a rapid and efficient method for the accurate diagnosis of paulownia witches'-broom disease.

並列關鍵字

paulownia witches'-broom ； phytoplasma ； nonradioactive DNA probe ； polymerase chain reaction