可同時檢測多種百香果病毒之多目標型增幅及晶片檢測系統

Development of multiplex RT-PCR and biochip detection systems for simultaneous detection of passionfruits viruses

黃莉真(Li-Chen Huang)

指導教授 ： 張清安

朝陽科技大學/理工學院/應用化學系生化科技碩博士班/碩士(2014年)

摘要

台農一號百香果為目前台灣最主流之栽培品種，由於此品種乃黃色種與紫色種百香果一代雜交之後代，因此其種苗必須以阡插或稼接等無性繁殖方式才能維持品種特性。民國70年間台灣百香果曾經因為病毒病的感染蔓延全台，幾乎摧毀了當時的百香果果汁產業。因此目前百香果種苗之生產必須建立無病毒感染之供穗園，定期檢測確定無病毒感染後方能採穗嫁接繁殖苗木。近年來百香果除台灣外亦極受東南亞國家及中國大陸地區消費市場歡迎，外銷種苗需求量快速增加，因此病毒檢測也有穩定之需求。過去本研究室建立無病毒百香果苗繁殖體系時曾發展可檢測此三種病毒之ELISA技術，但過程中必須分別進行至少三次檢測，程序繁複成本也較高。本研究乃基於過去發展生物晶片檢測法之經驗，希望利用其可以同步檢測多種病毒之優勢，加上檢測標的為病毒核酸特別適合偵測初期感染之樣品。過去台灣已記錄之百香果病毒種類共有三種，包括二種Potyvirus屬病毒；百香果斑紋病毒(Passionfruit mottle virus, PaMV)及百香果木質化病毒(Passionfruit woodiness, PWV)，再加上胡瓜嵌紋病毒 (Cucumber mosaic virus, CMV)。根據最新國際病毒分類委員會(ICTV)及澳洲發表的資料，台灣過去所稱之PWV經序列比對後發現與日本所發表之East Asian passiflora virus (EAPV)在鞘蛋白基因(CP)上幾近相同，故認定台灣所稱之PWV乃EAPV之同種異名。本研究重新進行過去長期保存之PWV及PaMV分離株 CP基因之定序分析。比對後確認台灣PWV分離株與EAPV應屬同種病毒。甚至PaMV之CP序列與EAPV之相同度仍高於80%，依ICTV之分類標準PaMV也應歸屬於EAPV之同種病毒。然而進一步分析發現日本與烏干達所發表之18個EAPV分離株序列中事實上有3種與台灣PaMV間有高達95%以上的相同度，而此三種與其他EAPV及台灣之PWV分離株在CP基因序列相同度上僅有81-85%。此一現象經PHYLIP軟體分析演化親緣樹後亦顯示現今所有登錄為EAPV之分離株應存在二大族群，族群間在CP序列相同度上約有10-15%之差異。以PaMV與EAPV相較，兩者間CP胺基酸序列相同度為84%；兩者之CP與NIb基因間之切位不同；兩者在CP基因之N端序列上有極大之差異；加上3’端非轉譯區核苷酸相同度僅為85%。此種差異吾等認為應可將此二族群歸納同種病毒之不同亞種(subspecies)。另外本研究已完成可同時檢測EAPV及PaMV等potyviruses及CMV之多目標型RT-PCR增幅及生物晶片雜合反應系統之開發，利用各自針對EAPV及PaMV之專一性引子對，可分別穩定增幅出613 bp及735 bp核酸產物，加上過去本實驗室已開發可穩定增幅CMV產出511 bp產物及可廣效增幅所有potyviruses病毒增幅出374 bp產物之potyvirus generic primer等四組引子對同時組合運作下，證實可穩定增幅單一或複合感染之百香果樣品，產生相對應之產物。此等產物再經生物晶片系統反應後，則可與本研究所設計之寡核酸探針產生雜合反應，判別感染病毒之種類。另外由於近年來台灣百香果又發現遭受兩種粉蝨傳播之begomoviruses感染，分別為大戟捲葉病毒 (Euphorbia leaf curl virus, EuLCV) 及木瓜捲葉廣東病毒 (Papaya leaf curl Guangdong virus, PaLCuGDV)，本研究也已完成可同時增幅此二病毒之一組專一性引子對之設計與測試，可分別針對上述兩病毒穩定增幅出608 bp之核酸產物。接著分別設計兩組可區別此二病毒增幅產物之專一性探針，在晶片雜合系統下證實可鑑別此二雙生病毒之感染。利用上述所發展之晶片系統檢測由南投埔里、草屯、苗栗南庄田美、苗栗竹南等地區所採集之百香果樣品，並與RT-PCR檢測結果比對，證實能獲得穩定相符之檢測結果。

關鍵字

百香果病毒

並列摘要

Passionfruit cultivar, Tainung No. 1, has been the dominant passionfruit cultivar in Taiwan for more than 30 years since it was officially released in 1980. Due to its F1 hybrid nature between Passiflora edulis and P. edulis f. flavicarpa, this cultivar can only be propagated by vegetative means to maintain its horticultural traits. Unfortunately, this unique propagation method allowed viral pathogens extensively disseminated to the propagated materials. In 1980s, virus diseases almost devastated the passionfruit juice production industry in Taiwan. Nowadays, virus disease of passionfruit in Taiwan was successfully managed by an integrated strategy of planting virus-tested growing materials and field control program. As a result, Taiwan’s virus-tested passionfruit growing materials have been highly popular in domestic market; they were also exported to foreign countries including Vietnam, Thailand and mainland China. To our knowledge, the demand of virus-tested passionfruit materials from abroad these years has kept increasing thus urged a consistent need of virus testing kits. In the past 30 years, ELISA technique was routinely applied in passionfruit virus detection and certification of virus free growing materials. These serological techniques normally targeted individual virus separately by specific antibodies, therefore the more viruses infected a crop the more tests were needed that resulting higher cost in labor and consumables. This study attempted to develop a multiplex detection system for passionfruit viruses to increase the detection efficiency and cost effectiveness. Based on literature, there have been three viruses, including Cucumber mosaic virus (CMV), and two potyviruses, i.e. Passionfruit woodiness virus (PWV) and Passionfruit mottle virus (PaMV), reported to infect passionfruit in Taiwan. However, in 2011 a report published by Australian working group suggested that the Taiwanese PWV isolate should be renamed as East Asian passiflora virus (EAPV) for both viruses share more than 80% identities in amino acid sequence of their coat protein (CP) gene. In addition, the Taiwanese PWV and EAPV originally published in Japan were both different from the original PWV isolate published in Australia, sharing less than 70% amino acid sequence identities in their CP genes. In the first part of our study, we cloned and sequenced again the CP gene of the original PWV isolate which has been preserved in our lab for more than 30 years. We also cloned and sequenced CP gene of PaMV, the second passionfruit potyvirus found in Taiwan, to study their authentic taxonomic status. Our comparative studies have confirmed that indeed our PWV isolate should be considered as the same species of EAPV for their more than 96% of amino acid sequence identities in CP genes. Surprisingly, our PaMV should also be regarded as EAPV for their CP gene sequence identities also higher than 80%. However, we have found that there were three EAPV isolates reported from Japan shared more than 98% CP sequence identities with our PaMV and these isolates all shared only 81-85% identities with the other 15 EAPV isolates from Japan and Uganda. This result implicated that there actually were two clades of sequences among the documented EAPV isolates in the GenBank. To confirm this postulation, a phylogenetic analyses using PHYLIP software were conducted and the result supported our speculation. Actually, not only the CP gene sequences had about 10-15% of differences between these two groups of EAPV; they were found also different in their digestion site between NIb and CP gene. Moreover, the nucleotide sequences identities in the 3’ non-translated region (3’-NTR) between these two EAPV groups were only about 85%, which was apparently lower than those normally found between isolates or strains of the same potyvirus species. This information implemented that Taiwanese PaMV and at least the other three isolates of EAPV probably represented a separate subspecies of EAPV. Secondly, we have successfully developed a multiplex RT-PCR amplification and biochip hybridization system for the aforementioned passionfruit viruses. By combining two primer pairs specific to PaMV and EAPV that could amplify 735 bp and 613 bp products, respectively; a CMV specific primer amplifying 511 bp product and a Potyvirus-generic primer producing 374 bp DNA fragment, a RT-PCR amplification system was developed and confirmed to be feasible for multiplex detection of PaMV, EAPV, CMV and other possible potyvirus, either single or mixed infection, in passionfruit. We also designed specific oligonucleotide fragments complimentary to each PCR amplicon from these passionfruit viruses and implanted on the surface of biochip as probes to react and differentiate the identities of PCR products. Besides the three aforementioned viruses, there were two begomoviruses, i.e. Euphorbia leaf curl virus (EuLCV) and Papaya leaf curl Guangdong virus (PaLCuGDV) reported in 2012 infecting passionfruit in Taiwan. To detect these two newly emerged viruses, a common primer pair amplifying both a 608 bp product in PCR from these two begomoviruses was developed. Two oligonucleotide probes respectively specific to the 608 bp PCR product from EuLCV and PaLCuGDV were designed and selected to distinguish infection of these two begomoviruses in biochip hybridization system. Seventeen field collected passionfruit samples were tested the feasibility of these two detection system and confirmed that both multiplex PCR amplification and biochip hybridization could gave consistent detection results.

並列關鍵字

passionfruits viruses

參考文獻

何宇倫。茄科重要病毒鑑定晶片之研發與應用。碩士論文，國立台灣大學植物病理與微生物研究所。台北 (2007) 。

林靜宜、蔡文錫、古新梅、詹富智。轉基因策略發展雙生病毒抗性植物之現況及展望。植病會刊18: 185-200 (2009)。

趙佳鴻、陳慶忠、黃秋維。瓜類兩種馬鈴薯Y群病毒之傳播與生態學研究。植病會刊3:84-90 (1994)。

張清安、林瑩達。無病毒百香果苗栽植後之再感染生態及其防治病毒病效果評估。 植物病理學會刊1:140-146 (1992) 。

黃尚仁。可同時檢測三種病毒之香蕉生物晶片系統開發。碩士論文，朝陽科技大學生化科技研究所，台中 (2012) 。