木瓜輪點病毒之Real-Time RT-PCR定量偵測技術之研發與應用

Abstract

木瓜輪點病（papaya ringspot）是木瓜最重要病害之一，嚴重影響木瓜產業。台灣於1975年發現後即迅速傳播至全省各木瓜主要產地。此病由木瓜輪點病毒（Papaya ringspot virus, PRSV）所引起。目前在台灣的PRSV又可依據在木瓜上引起病徵的不同，再細分成各種不同系統（strains），包括可引起葉片產生嚴重嵌紋病徵的嚴重嵌紋系統（severe mottling, SM strain）、造成葉片嚴重嵌紋且畸形扭曲的畸形系統（deformation, DF strain），以及造成葉片嚴重嵌紋且伴隨有壞疽病斑的嚴重嵌紋壞疽系統（severe mottling with necrosis, SMN strain），SMN系統更會在冷熱交替之際造成快速萎凋的病徵。過去在台灣木瓜栽植區所見到的病徵大多是以SM系統為主。但是，近年來台灣田間罹病木瓜多被DF與SMN系統漸漸取代。以本研究室所保存之純系病毒株，利用5’ RACE與3’ RACE的兩端定序，完成此三系統的全長定序，其核酸序列總長度皆為10326 nt。根據全長序列分析結果顯示出SM與DF的核酸序列相似度達97.3％，而SMN與它們的相似度分別為97.0％與96.8％。此三系統的各基因核酸序列相似度約在95∼100％之間，唯有在P1基因的核酸序列相似度低於95％，並發現他們在5’UTR（untranslated region）相似度更低，僅91.8∼92.9％。進一步比對此三系統與其他國內外PRSV分離株的核酸與胺基酸序列，發現SM與DF系統親源關係最近似，SMN系統次之，而三系統與台灣永康分離株歸屬同一關係群，而與國外分離株親源關係較低。目前對三個不同的PRSV系統，已研發出RT-PCR快速偵測法，以改過的TRIzol reagent方法，可以有效減少抽取核酸所花費時間，且利用全長序列分析結果設計另一通用性引子對PRSV-829，為一改良過的引子對，可提供更高專一性與正確性的PRSV偵測，尤其可避免對轉基因木瓜(植入PRSV的鞘蛋白基因)產生的偽陽性反應。為了更進一步對不同PRSV系統在木瓜寄主體內做定量追蹤，將real-time RT-PCR技術應用於PRSV的偵測與消長動態分析。在本論文，將SYBR Green與TaqMan primer/probe兩種方法同步進行偵測設計。在SYBR Green方法方面，利用5’UTR與部分P1基因序列設計出通用性引子對PRSV-159，但結果不如預期，僅對SM與SMN兩系統有預期的螢光值增加曲線，DF系統則無；然而此方法對SM系統敏感度極佳，即使模版量僅約0.2 ng都可利用此法測得，做成的相對定量曲線也符合預期，顯示SYBR Green法在條件合適狀況下，仍可獲得對SM系統相當好的偵測效果。在TaqMan primer/probe方法方面，利用鞘蛋白基因（CP gene）中的保守性區域設計出三系統皆可通用的TaqMan primer/probe套組（命名為TP-PRSV），預計增幅長度約95 bp，並在其中設計一條16 nt的TaqMan probe以進行螢光偵測，實驗證實定量偵測的效果良好，且其敏感度更勝SYBR Green系統。進一步，也利用三系統在P1基因序列上的相異處，另設計三套具系統特異性的TaqMan primer/probe套組，分別命名為TP-DF，TP-SM及TP-SMN，實驗結果也顯示三個病毒系統的專一性定量偵測效果相當良好。為了追蹤PRSV之不同系統在接種後，於木瓜寄主體內繁殖量的變化情形，本論文挑選目前病徵呈田間優勢的DF系統與極具威脅性的SMN系統，單獨或混合接種於番木瓜之紅妃品系上，再以上述研發的real-time RT-PCR技術追蹤PRSV在寄主體內之增殖情況。結果顯示單獨感染DF系統時，在第10天病毒訊號即明顯出現，隨後增殖曲線開始攀升至第16天達第一個高峰，第16~22天呈現停滯，但第24天後病毒訊號就速攀第二高峰而進入高原期；單獨感染SMN系統時，在第10天即開始測得明顯訊號，隨即呈現線性上升，至第18天後病毒訊號可達高峰。至於複合感染DF與SMN系統的木瓜植株，DF系統直到第16天才測得第一個病毒訊號，至第18天訊號迅速上升，至第22天病毒訊號達高峰；SMN系統在第18天以前幾乎測不到病毒訊號，至第18天測得第一個訊號，第22天後訊號迅速達到高峰。因此，利用此套real-time RT-PCR不但可以同步定量目標病毒系統，而其應用性也相當廣，不但可以研究不同木瓜輪點病毒系統，在寄主體內的增殖動態，未來進一步建立real-time RT-PCR的標準定量曲線，更可將此技術發展至PRSV之絕對定量，以供木瓜輪點病的發病生態相關研究使用。

Papaya ringspot is one of the most destructive diseases of papaya and it is a limiting factor for papaya industry. This disease first occurred in Taiwan in 1975, and it has spreading widely throughout all of papaya-cultivated areas in Taiwan. It is caused by Papaya ringspot virus (PRSV), which belongs to Potyvirus, Potyviridae. According to the different symptoms in papaya hosts, PRSV was divided into three major strains including SM (severe mottling), DF (severe mottling with leaf-deformation) and SMN (severe mottling with necrosis and quick decline) strains. SM was a predominant strain of PRSV in the field of Taiwan several years ago. However, DF and SMN have recently become newly rising dominant strains. Almost full-length of genomic sequences of three PRSV strains were determined in the previous study. Continuous sequencing was conducted in this study. The complete nucleotide sequences (10326 bases) of three strains were determined by application of 5’ RACE and 3’ RACE systems. They encode a polyprotein of 3344 amino acids with a 5’ untranslated region of 85 nucleotides and a 3’ untranslated region of 209 nucleotides. The results of alignment of full genomic nucleotide sequences demonstrated that SM is 97.3% identical to DF, and SMN is 97.0% and 96.8% identical to SM and DF. Most genes in the PRSV genome among the three strains are 95~100% homologous, but the P1 gene and 5’ UTR region are about 95% and 92% homologous among three different strains, respectively. Phylogenic analysis of various international PRSV isolates revealed that SM, DF and SMN are close to the Yung-Kang isolate (another PRSV isolate from Taiwan), but far away from the Thailand and Hawaii isolates. The rapid assay based on reverse transcription-polymerase chain reaction (RT-PCR) with the primer pair PRSV-857 was developed for the detection of PRSV in previous study. Another primer pair PRSV-829 was devised in our study, and it has been proven to achieve better specificity and sensitivity in PRSV detections. In addition, the real-time RT-PCR technology was applied for quantitative monitoring of different PRSV strains in papaya hosts. Both “SYBR Green” and “TaqMan primer/probe” methods were adopted to develop the quantitative detection of PRSV with real-time RT-PCR, and “TaqMan primer/probe” method obtained more satisfactory results. A TaqMan primer/probe combination (named TP-PRSV), selected from the conserved regions of coat protein gene, was designed for the common detections of PRSV. Three primer/probe kits (named TP-DF, TP-SM, and TP-SMN), selected from the variable regions of P1 gene, were also developed for the strain-specific quantitative detection of the DF, SM and SMN strain, respectively. Our devised real-time RT-PCR assays were further applied to monitor the virus multiplicative dynamics in papaya hosts in the inoculation tests with different PRSV strains. When the papaya hosts (cultivar FR) were inoculated with the DF strain, PRSV could be detected 10 days after inoculation. The replication curve (DF) was increasing linearly from the 10th to 16th day, stationary from the 16th to 22th day, and reaching to the peak 24 days after inoculation. When the papayas were inoculated with the SMN strain, PRSV could be detected 10 days after inoculation. The replication curve (SMN) was increasing linearly from the 10th to 18th day, and reaching to the peak 18 days after inoculation. When the papayas were inoculated with both DF and SMN strains, DF could be detected 16 days after inoculation. The replication curve (DF) was increasing linearly from the 16th to 22th day, and reaching to the peak 22 days after inoculation. On the other hand, SMN could be detected 18 days after inoculation. The replication curve (SMN) was increasing linearly from the 18th to 22th day, and reaching to the peak 22 days after inoculation. The development of real-time RT-PCR assays of PRSV is helpful to the ecological studies of papaya ringspot disease.