台灣茄科類病毒快速診斷方法之建立與應用

The development and application of a fast simultaneous detection to Solanaceae viroids

廖彥博(Yen-Po Liao)

指導教授 ： 沈湯龍

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201603378

摘要

植物類病毒為目前已知最小的植物病原，由一段無編碼蛋白的單股環狀RNA片段所組成，大小僅有249至401個核酸，但可藉由其序列互補的特性形成二級及三級結構。類病毒會造成多種植物病害，包含草本和木本作物及園藝植物，降低作物的產量。目前在茄科作物上已有多種類病毒被報導，包含馬鈴薯紡錘形塊莖類病毒(Potato spindle tuber viroid，PSTVd)、蕃茄褪綠矮化類病毒(Tomato chlorotic dwarf viroid，TCDVd)、番椒小果類病毒(Pepper chat fruit viroid，PCFVd)、番茄頂縮類病毒(Tomato apical stunt viroid，TASVd)、蕃茄植株雄化類病毒(Tomato planta macho viroid，TPMVd)、金魚花潛隱類病毒(Columnea latent viroid，CLVd)。雖然上述類病毒在台灣均尚未被報導過，但皆已被列為國際重要植物作物檢測項目。因此，建立一套快速且有效的診斷方法是重要且必要的。本研究目前已可在同一條件下快速同步偵測六種類病毒，並且已嘗試應用在台灣番茄種子進出口之檢測上，目前已檢測來自不同國家的45批種子，除此之外，本研究也藉由設計增幅不同大小的引子對，進行檢測方法之改良，以方便之後進行多重聚合酶連鎖反應(Multiplex PCR)使用，並大幅減少相關試劑的使用,目前已設計出CLVd-314F/R 並將其搭配先前設計之TASVd, TPMVd引子達到同時偵測三種類病毒之多重聚合酶連鎖反應。

關鍵字

茄科類病毒 ； 反轉錄聚合酶連鎖反應 ； 多重聚合酶連鎖反應

並列摘要

Viroids are circular non-coding single-stranded RNAs (249-401 nt) and could form secondary and tertiary structure by its special high complementary sequences. They are known as the smallest plant pathogen, and can infect wide variety of crops, including woody, herbaceous and horticultural plants. Until now, there are several viroids reported on the Solanaceae plants, including Potato spindle tuber viroid (PSTVd), Tomato chlorotic dwarf viroid (TCDVd), Pepper chat fruit viroid (PCFVd), Tomato apical stunt viroid (TASVd), Tomato planta macho viroid (TPMVd), Columnea latent viroid (CLVd). Though all the viroids above are not reported in Taiwan before, they have been listed as quarantine pests in the world. Thus, it is important and necessary to build up an efficient and quick diagnosis method to detect these viroids. This study has built a fast simultaneous detection for six Solanaceae viroids and the detection technique has been used on the import tomato seed viroid detection. Now, we have already detected 45 lots tomato seed from different countries including some of the Solanaceae viroid epidemic area. In addition, we also design several primer pairs of viroids for future multiplex RT-PCR, though these primer pairs still on test process, it would greatly reduce the amount of detection kit if the multiplex RT-PCR succeds. In this work, we have designed the primer pair CLVd-314 F/R, and combine the futher TASVd and TPMVd primer pairs to achieve three viroid multiplex PCR.

並列關鍵字

Solanaceae viroid ； reverse transcription-PCR (RT-PCR) ； Multiplex polymerase chain reaction

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