Reverse dot-blot hybridization method for identification of nine potyviruses

Potyviridae科中的Potyvirus屬，是植物病毒中最大的一屬；目前所知的種類近200種，對人類的糧食及經濟作物造成的危害頗大。因為馬鈴薯Y屬病毒的種類繁多，因此發展出一套能快速檢測並區別各種病毒的鑑定方法，將有助於植物檢疫與防疫工作。生物晶片是21世紀的生物技術重點產業，最大特點就是在同一片晶片上，可同時處理多項訊息。本論文希望利用晶片和potyvirus的特性，建構potyvirus的鑑定晶片系統。晶片上的探針分為cDNA探針和寡核苷酸探針兩種，其序列位置均落在potyvirus的NIb基因3’端和CP基因5’端之間。標的物是以potyvirus的cDNA株或感染potyvirus的植物全RNA，與potyvirus的廣效性引子對進行PCR或RT-PCR反應時，加以標定得之。將cDNA探針固定在尼龍膜上後，與標的物進行逆墨點雜合反應，結果發現所製備的cDNA探針專一性很好。由此所建構的cDNA晶片可以正確地鑑定出BaRMV、PRSV、PVA、PVY、TuMV、 ZaMV和ZYMV等八種病毒單獨感染的植物樣品。此外，cDNA晶片也可以正確的鑑定出兩種或三種複合感染的植株。而為了確定寡核苷酸探針的最適長度和最佳雜合條件，我們根據ZaMV和ZYMV的序列，設計不同長度的寡核苷酸探針，結果以50 mer的效果最好。故針對不同的病毒，設計50 mer寡核苷酸探針，固定在尼龍膜上，與標的物進行逆墨點雜合反應，結果發現這些探針的專一性相當良好。由此所建構的寡核苷酸晶片可以成它a鑑定出上述八種病毒；同時也可以成它a鑑定複合感染的樣品。檢視兩種晶片的鑑定結果，雖然cDNA探針靈敏度較寡核苷酸探針為高，但是寡核苷酸探針擁有不須具備病毒cDNA株即可製備的優點。日後若有新種病毒出現或是原有病毒序列產生較大變異，則只需加入新的寡核苷酸探針即可維持鑑定晶片系統的完整性，應該是未來發展的趨勢。The genus Potyvirus in the family Potyviridae is the largest genus of plant viruses and can infect a wide range of crop plants. Because there are about 200 species in the genus, it is important to develop a rapid identification system for potyviruses to support the tasks of plant quarantine and inspection. Biochip has become an increasing popular tool of biotechnology industry at 21 century, because it can deal with hundreds to thousands information at the same time. The aim of this thesis is to develop a potyvirus identification chip based on the traits of potyviruses and biochip. Two kinds of probes, cDNA and oligonucleotide probes were prepared, and their sequences derived from the 3’end of the NIb gene and the 5’end of the CP gene. The DIG-labeled targets were prepared from viral cDNA clones or total RNA of infected tissues by means of PCR or RT-PCR with potyvirus degenerate primers. After cDNA probes immobilized onto the nylon membrane, the targets were tested by reverse dot-blot hybridization. The results indicated that our cDNA probes had high specificity to the targets. When using our cDNA chip to test the targets derived from plant total RNAs, it could successfully identify eight potyviruses including BaRMV, PRSV, PVA, PVY, TuMV, ZaMV and ZYMV. Moreover, the cDNA chip could also identify mix infection plants with two or three kinds of potyviruses. In order to define the optimal length of oligonucleotide probe, probes with different lengths of ZaMV and ZYMV sequence were designed, and the effectiveness of probes were compared. The results revealed that the 50-mer probe of specific virus sequence gave constant positive results and thus was used for further experimental design. The specificity of 50-mer oligonucleotide probes of potyvirus tested by reverse dot-blot hybridization was satisfying. When using our oligonucleotide chip to test the targets derived from plant total RNAs, it could successfully identify the aforementioned eight potyviruses and also mix-infection samples. Comparing the results of two kinds of potyvirus chips, cDNA probes showed better sensitivity than oligonucleotide probes, but oligonucleotide probes had the advantage of preparation without viral cDNA clones. If new viruses appear or the existing viruses produce quite a few mutations, we only need to add new oligonucleotide probes to maintain the completeness of the identification chip. Therefore, oligonucleotide chip is better choice than cDNA chip and will be a novel way of rapid identification for potyviruses in the future.中文摘要---1 英文摘要---2 第一章 前人研究---4 第一節 馬鈴薯Y屬病毒之簡介---4 第二節 生物晶片之簡介---8 第三節 逆墨點雜合反應---12 第二章 Potyvirus鑑定晶片系統之研發與建立---13 前言---13 材料與方法---14 2.1 實驗植物---14 2.2 病毒來源---16 2.3 病徵描述---16 2.4 植物全 RNA 之抽取---19 2.5 Potyvirus 廣效性引子對與種專一性引子對之設計---19 2.6 反轉錄反應---20 2.7 聚合酶鏈鎖反應---20 2.8 轉型試驗---20 2.9 質體DNA之小量製備---21 2.10 cDNA探針的製備---21 2.11 寡核苷酸探針的設計---22 2.12 標的物之製備---23 2.13 逆墨點雜合反應---23 第三章 Potyvirus鑑定晶片系統之試驗結果---25 3.1 Potyvirus廣效性引子對之檢測--- 25 3.2 cDNA探針的製備結果---25 3.3 cDNA探針的專一性---26 3.4 cDNA晶片的鑑定結果---26 3.5 寡核苷酸探針長度的試驗結果---27 3.6 寡核苷酸探針的專一性---27 3.7 寡核苷酸晶片的鑑定結果---28 第四章 討論---29 參考文獻---35 圖表---4