紅龍果potexviruses病毒快速檢測方法與磁性奈米粒子療法之開發及應用

Abstract

紅龍果 (dragon fruit; pitaya) 屬於仙人掌科 (Cactaceae)、三角柱屬 (Hylocereus spp.) 的多年生攀緣性肉質植物，由於紅龍果外觀討喜，果實風味佳，且具高營養價值，加上目前台灣積極推廣紅龍果的外銷，使紅龍果成為極具市場經濟潛力的熱帶果樹。台灣目前多以扦插方式進行繁殖，故若作為扦插原的紅龍果母本為帶病毒植株，即會使病毒在田間快速擴散。根據前人研究，Potexvirus屬的仙人掌X病毒 (Cactus virus X, CVX)為台灣第一個被報導可以感染紅龍果的病毒，2006年本實驗室於陽明山觀光果園的紅龍果發現除了CVX外，尚有在台灣未曾報導過的蟹爪蘭X病毒 (Zygocactus virus X, ZyVX)，以及一種新的potexvirus病毒，命名為紅龍果X病毒 (Pitaya virus X, PiVX)。由田間調查顯示這三種病毒常感染紅龍果，而Potexvirus屬病毒也是目前唯一被報導會感染紅龍果的病毒屬。本研究利用羧基 (-COOH) 修飾之四氧化三鐵 (Fe3O4) 磁性奈米粒子，開發出一套針對紅龍果中三種potexvirus病毒之快速檢測方法，命名為magnetic nanoparticle-capture reverse transcription-polymerase chain reaction (MNC RT-PCR)，以利往後病毒檢測。也藉由此檢測方法對其他同為仙人掌科之十種常見觀賞仙人掌：蟹爪蘭、白烏帽子、金烏帽子、紅牡丹、金鯱、兜丸、劍戀玉、岩石獅子、六角柱仙人掌與神仙堡進行檢測，以及對多處台灣紅龍果產區，包含宜蘭、彰化、台東與屏東之紅龍果進行田間調查，分析各地區紅龍果植株的病毒感染率與分布情形。根據目前調查結果顯示，發現蟹爪蘭遭受CVX以及ZyVX感染，將這兩種病毒基因體片段選殖並定序後，於NCBI上進行BLASTn與BLASTx分析。結果發現感染蟹爪蘭的ZyVX與紅龍果中ZyVX-P39在核酸序列以及RdRp與TGB1-3的胺基酸序列相同度皆達95%以上。而CVX的部分RdRp序列與前人在關西果園中發現之CVX-Hu序列相同度達95%，但CP胺基酸序列卻是與韓國之CVX-Jeju3分離株較相似，序列相同度高達99%。本次調查證明此三種potexvirus病毒也存在於紅龍果外的其他觀賞仙人掌上。而田間調查部分，由檢測結果可知除台中農試所外，田間已不存在健康紅龍果植株，推斷是由於使用帶病毒扦插枝條，加上長期使用未經消毒之工具進行連續採收與修枝，導致病毒以機械傳播方式擴散至全園區。故本研究也擬開發一套奈米粒子療法，同樣利用修飾 –COOH 之Fe3O4磁性奈米粒子作為主體，以具有專一辨識性的單股寡核苷酸片段與核糖核酸酶H (RNase H) 進行修飾，藉由對三種potexvirus RNA的專一性雜合，使磁性奈米粒子表面上鄰近之RNase H能夠截切目標病毒RNA受雜合之複製相關重要區域，以此降低受感染紅龍果中的potexviruses病毒複製能力，達到去除病毒的目的。奈米療法部分已完成辨識性寡核苷酸片段之選定與粒子修飾條件測試，將合成之粒子命名為Nanozyme，並已進行初步生體外去病毒RNA試驗。目前所使用之Nanozyme確實具有專一性辨識能力，但在裁切病毒RNA能力上效率不顯著，仍需進行實驗條件的測試與改良。

Pitaya, also called dragon fruit, which is a kind of perennial climbing succulent plant belongs to the genus Hylocereus in the family of Cactaceae. Because of its charming, delicious fruit with high nutrition and the export policy of government, pitaya has become an important tropical fruit crop in Taiwan. Pitaya is mainly propagated by cutting, so if mother plant is infected with viruses, disease can be spread easily. According to the previous studies, Cactus virus X (CVX), belonging to the genus Potexvirus, is the first pitaya-infecting virus reported in Taiwan. The other two potexviruses, Zygocactus virus X (ZyVX), first reported in Taiwan, and Pitaya virus X (PiVX), a new species of potexvirus, were first identified by our laboratory in the Yanmingshan orchard in 2006. The previous field survey indicated that these three potexviruses infect pitaya frequently, and so far Potexvirus genus is the only one virus genus reported to infect pitaya plants. In this study, a rapid detection method, named magnetic nanoparticle-capture reverse transcription-polymerase chain reaction (MNC RT-PCR), using Fe3O4 magnetic nanoparticles modified with –COOH for detecting the pitaya-infecting potexviruses was developed. Ten common ornamental Cactaceae plants, including Opuntia microdasys v. albispina Fobe., Opuntia microdasys, Gymnocalycium mihanovichii var. friedrichii, Echinocactus grusonii, Astrophytum asterias Lem., Echinofossulocactus kellerianus Krainz, Cereus peruvianus f. monstrosus, Cereus peruvianus Mill. and Cereus 'Fairy Castle', were tested for virus infection by MNC RT-PCR. Pitaya plants from several production areas in Taiwan, including Ilan, Changhua, Taitung, Pingtung and Taichung, were also detected by MNC RT-PCR to investigate the infection rate and distribution of potexviruses. The results of field survey revealed that zygocactus can be infected by CVX and ZyVX, and these three potexviruses can infect other Cactaceae plants. After cloning and sequencing the fragments of the two potexviruses from zygocactus and BLAST in NCBI database, it shows that the nucleotide sequence identity and amino acid sequence identities of RdRp and TGB1-3 ORF are above 95% between zygocactus ZyVX and pitaya ZyVX-P39. The nucleotide sequence identity of partial RdRp is 95% between zygocactus CVX and pitaya CVX-Hu, whereas CP amino acid sequence of zygocactus CVX is similar to CVX-Jeju3 from Korea with 99% identity. The field survey of pitaya shows that healthy pitaya plants could not discovered in the field except pitaya samples collected from Taichung Agricultural Research Institution. It may be that farmers use potexvirus-infected cuttings as propagules, and mechanical inoculation through contaminated tools cutting stems or harvesting fruits successively without disinfection. Due to the high infection rate of potexvirus in pitaya, the other target in this thesis is to develop a magnetic nanoparticle therapy by using Fe3O4–COOH modified with specific single strand oligonucleotides and Ribonuclease H (RNase H). When the target potexviruses are hybridized with specific oligonucleotides, the near RNase H can degrade hybridized viral RNA, to reduce the replication ability of target potexviruses. So far the sequences of specific identification oligonucleotides were determined, and the modification condition of Fe3O4–COOH, named the modified nanoparticle Nanozyme, was tested. The preliminary efficiency test in vitro indicated Nanozyme shows its specificity to recognize target potexviruses, but its degradation efficiency to target fragment is not obvious.