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Development of Techniques for Rapid Identification of Pest Organisms on Animals and Plants
|關鍵字:||植物保護類;應用研究;Vector-borne Zoonoses;節肢動物媒介人畜共通傳染病;診斷試劑;石英震盪器;奈米科技;墨點酵素連結免疫吸附法;分子鑑定;鑑別診斷;序列特徵化增幅區域;Diagnotic Kits;QCM;Nanotechnology;Dot-ELISA;Molecular Identification;Discriminating Diagnosis;Sequence Characterized Amplified Regions||摘要:||
1. Identification techniques and information compilation of arthropod vectors transmitting important infectious diseases of animals and man: The objective of this project is to collect, compile and analyze all the domestic and international research information regarding vector-borne infectious diseases (including zoonoses) and their transmission arthropod vectors. A book with a title of “Introduction to arthropod vectors and the infectious diseases they transmitted” will be published by the Bureau of Animal and Plant Health Inspection and Quarantine timely. This book will be beneficial to the professional of all related field. 2. Development and application of the integrated diagnostic kit for citrus systemic diseases: The systemic pathogens such as the bacteria causing citrus huanglongbing, Citrus tristeza virus (CTV), Citrus tatter leaf virus (CTLV) and Citrus exocortis viroid (CEVd) are predominant and destructive to citrus industry in Taiwan. These pathogens are mainly transmitted by vegetative propagation. Establishment of pathogen-free nursery systems and development of rapid and sensitive diagnostic probes are considered to be the most important methods for the pest control. This research project is dedicated to develop new diagnostic probes and improve several devised diagnostic methods using immunological and molecular techniques. An integrated diagnostic kit will be produced through combination of all our devised detection methods for these citrus systemic pathogens. This kit will be helpful to the inspection and management of the citrus pests, and it is worthy of international patent for world-wide extension. 3. Development nano-particle-sensitized QCM sensor for the real-time diagnosis and identification of pathogens in animals and plants: Utilizing gold nanoparticle binding with virus to increase virus weight, overcome detection limit and enhance detection response. This project will carry out the following important works this year: 1. Preparation of Gold nanoparticle; 2. To obtain and prepare sample; 3. To combine Gold nanoparticle and virus; 4. To develop mechanisms of sample's verifications and tests. 4. Development of techniques for rapid discrimination of various insect pests in imported grain: The goal of this project is to develop species-specific PCR primers for rapid discrimination of insect pests in imported grain. The approach will be founded on using random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Firstly, to compare the RAPD-PCR profiles of 3 major lepidopterous pests of grains, i.e. Plodia interpunctella H., Sitotroga cerealella O., and Cadra cautella; secondly, to isolate the species-specific amplicons from the gel and to further clone and sequence; thirdly, to design specific PCR primers based on the sequence; finally, to test the specificity and stability of PCR reactions with the designed primers. By the end of the research, we expect to develop 2-3 pairs of species-specific PCR primers for rapid species discrimination of each of these 3 pests. Furthermore, we also expect that this method is able to expend to be used on quarantine inspection of imported grain and to avoid the possible invasion of these pests. 5. Development of Molecular Diagnostic method for the rapid differentiation of Baylisascaris procyonis and Toxocara canis eggs: Baylisascaris procyonis is commonly found in raccoon and has been recognized as an emerging helmintic zoonosis in recent years. When B. procyonis embryonated eggs are ingested by a host other than the raccoon, larva migrans ensues. In human, this larval can invade the brain and eye, causing severe disease and sometimes death. Clinical findings include eosinophilic meningoencephalitis, eosinophilic cardiac pseudotumor and chorioretinitis. B. procyonis can also infect dog, using it as an intermediate host as well as final host. The infected dog can also shed B. procyonis eggs, thus posing a diagnostic problem for those dogs that are infected with Toxocara canis. Apart from the size difference, B. procyonis eggs and T. canis eggs are quite morphologically similar. Thus, it is important to develop a molecular biological technique to differentiate the two types of eggs. We propose to develop a molecular diagnostic method, using PCR, for the rapid differentiation of B. procyonis and T. canis eggs. 6. Application of bio-Nanotechnology for diagnosis and detection of Lily mottled virus in lilium: In recent years, the development of molecular biology is fast. Now many kinds of nucleic acid probes and polymerase chain reaction (PCR) methods have been applied to identify and detect the plant pathogens because of the fast, uniqueness and sensitive characteristic of biotechnology. Although the sensitivity of PCR is up to 50-100 fg, but it still needs a professional technician for operation and takes approximately 6.5 hours, and its cost is expensive. So it is difficult for widely application on detection of pathogens. Therefore, a rapid, simple, and high sensitive technique with low expenses for detection of plant pathogens is necessary. The aim of the present study was to develop an ultra-sensitive and excellent photostability probe for detection of Lily mottle virus in Lilium by using bio-nanotechnology. 7. Development of Ehrlichiosis Serodiagnosis: Ehrlichiosis was Zoonosis in dog and human. E. canis and E. chaffeensis reside and replicates within the mononuclear cells, the clinic signs were included: weight loss, anemia, lymphadenomegaly, and anorexia. This study will utilize bioinformatics to predict the hydrophilic region (HR) of E. canis outer membrane protein Omp30, and then expressed the Omp30HR and NucA-Omp30HR in E. coli expression system. They will be purified and quantities with Ni+ column and ELISA, separately. These results enable us to utilize Omp30HR or NucA-Omp30HR for Dot-ELISA to clinic serodiagnosis of dog or human ehrlichiosis. We will develop the good, convenient, sensitive, fast and cheap Dot-ELISA kit, in order to remote the quarantine epidemic prevention plan of Zoonosis. 8. The identification and detection of brown rot pathogens on peach: Brown rot disease is one of the most disruptive diseases on stone fruits worldwide and has been reported in Taiwan. This disease can be caused by Monilinia fructicola, M. laxa, M. fructigena and Monilia polystroma. M. fructicola and M. laxa attacks mainly stone fruit, while M. fructigena attacks both stone and pome fruits but is found more frequently in the latter. Each brown rot pathogen of stone fruits has a different regulatory status. For example, M. fructigena is listed as a quarantine pathogen in the USA and in Australia. To understand the species of brown rot pathogens exist in peach orchards in Taiwan and to monitor the species of brown rot pathogens on imported fruits, a fast and accurate identification method to identify the species of brown rot pathogen needs to be established. In this study, we will isolate brown rot pathogens of peach from different peach growing areas in Taiwan and from imported stone fruits including cherry, peach and nectarine. The preliminary identification of those collected cultures will be performed based on the colony morphology, conidial size and sporulation type. A PCR based identification method with specific molecular markers will be developed to identify the species of collected brown rot pathogens. The genetic variation of brown rot pathogens within those collected from orchards in Taiwan and those from other countries will also be studied. 9. Application of liquid array for detection of avian Mycobacteriosis in zoo birds: Mycobacterium avium is pathogenic to most avian including domestic birds, pet birds, cage birds, wild birds, and zoo birds but most frequently infects domestic birds. It has also been reported in swine, rabbit, deer, cattle, lamb, and marten and is recognized as an important zoonosis. Histopathologically, multifocal nodules were observed in many organs. The goal of this study is to establish liquid array in combination with pathological examination, Ziehl-Neelsen staining, xMAP, and nucleotide sequencing to detect avian tuberculosis. 10. Development of immunoproteomics platforms to identify candidate antigens of classical swine fever virus for discriminating diagnosis: Classical swine fever (hog cholera) is a highly contagious disease of pigs and caused by classical swine fever virus (CSFV). The commonly used vaccine in Taiwan against hog cholera is an attenuated LPC strain of CSFV that made from type 820 CSFV. At least 141 CSFV strain have been isolated in the wild field of Taiwan. Base on their genomic sequences, all the CSFV can be divided into three genotypes; LPC strain belongs to genotype 1 and wild field CSFV belongs to genotype 2. Because LPC strain is different from wild field CSFV in the genotype and vaccination or infection pathways, and that may induce different antibody responses. In this plan, we would like to identify the differences of candidate antigens between two genotypes of CSFV by immunoproteomic technique, and then use to distinguish between LPC-vaccinated and wild CSFV infected pigs. Sera from pigs with LPC vaccine or wild field CSFV infections and control group of sera from health individuals will be analyzed for reactivity by western blot against LPC virus or wild field CSFV total proteins separated by 2-dimemional electrophoresis. We predict several immunogenic protein spot will be identified by immunoproteomic analysis, MALTI-TOF and LC-MS-MS. In the future, the candidate antigens will be developed to a rapid and easy diagnosis kit and use to discriminate between LPC vaccinated and wild CSFV infected pigs.
1. 媒介重要人畜傳染病之有害生物鑑定資訊與技術建立：本計畫主要收集並分析有關國內外媒介重要人、家畜及人畜共通傳染病節肢動物的研究資料，再加上這些傳染病基本資訊，邀集國內學術單位等專家撰稿審查，依進度出版「媒介重要人畜傳染病的有害生物-節肢動物篇」一書，以提供動植物防檢疫局和相關單位人員參考。使我國動物疫病防疫檢疫單位有完整的參考資料，對有害生物尤其是媒介傳播人畜傳染病生物媒介之鑑定編輯成冊以提供國內執行防檢疫人員參考。 2. 柑橘系統性病害整合性診斷試劑套組之開發與應用：台灣重要柑橘系統性病原包括黃龍病細菌、萎縮病毒(CTV)、破葉病毒(CTLV)與鱗砧次病毒(CEVd)等，這些病原經由帶病種苗普遍傳播而危害嚴重，生產無病種苗搭配敏感且正確的檢疫技術乃是防治這些病害的首要之道。本計畫以原本初步研發成功的柑橘病原檢疫技術為基礎，進一步再研發與改善，目標在於建立一套方便且快速的整合性偵測套組。具體工作項目包括： (1) 製備出柑橘破葉病毒之單元抗體； (2) 研發CEVd快速RT-PCR偵測技術； (3) 改良黃龍病細菌、CTV、CTLV之分子偵測法等。所得研究成果不但可供國內防檢疫之應用，更因這些病原皆為國際性重要病原，未來可申請專利而推展於國際。 3. 開發奈米粒子敏感化石英晶片感測器以即時診斷鑑定動植物有害生物：利用奈米金粒子(gold nanoparticle, GNP)和病毒結合，以增加病毒本身重量，以突破QCM的感測極限及增加偵測反應。本計畫今年將執行下列重要工作： (1) 製備金奈米粒子(Gold nanoparticle, GNP)； (2) 樣本取得及製備； (3) 奈米金粒子與病毒結合； (4) 樣本檢測驗證機制。 4. 進口大宗穀物害蟲之快速鑑定技術開發：本研究的主要目的在於利用聚合酶連鎖反應-逢機增幅多態型DNA (random amplified polymorphic DNA-polymerase chain reaction, RAPD-PCR)的方法，研發及尋求特定的DNA標誌，以供快速檢測與鑑定積穀害蟲之用。本計畫主要將先利用RAPD技術比對三種主要鱗翅目積穀害蟲，包括印度穀蛾(Plodia interpunctella H.)、麥蛾(Sitotroga cerealella O.)與粉斑螟蛾(Cadra cautella)等，尋求具種特異性的DNA片段，再進而依此片段之序列設計具有蟲種專一性的引子，並更進一步檢測其專一性與穩定性，以提高鑑定的準確度。預計本研究結束時，可建立利用PCR快速檢測上述三種積穀害蟲的方法，將可作為日後應用此技術以監測大宗進口穀物是否夾帶外來種之鱗翅目害蟲，進而有效地防堵外來害蟲種入侵的機會。 5. 開發分子生物診斷法來快速鑑定及識別浣熊蛔蟲與犬蛔蟲：浣熊蛔蟲主要寄生於浣熊，近幾年來被視為一種的新興疾病。當含有仔蟲的蟲卵被浣熊以外的宿主攝入時，會造成幼蟲移行症。當人被感染時，幼蟲可能移行至腦部及眼球而造成嚴重的臨床症狀甚至死亡。臨床症狀包括嗜酸性球性腦膜腦炎、嗜酸性球性心臟偽腫瘤及脈絡膜視網膜炎等。犬隻可擔任浣熊蛔蟲的中間宿主及終宿主。犬隻感染浣熊蛔蟲或犬蛔蟲時皆會排出蟲卵，因而造成鑑定上的困難。兩種蛔蟲之蟲卵的外觀除大小之差異外，在型態學上非常相似。因此開發分子生物診斷法來快速鑑定及識別有其重要性。本計畫將利用PCR法來建立快速診斷法鑑別浣熊蛔蟲及犬蛔蟲。 6. 生物奈米技術在百合斑駁病毒病害診斷與偵測之運用：近年來，分子生物學的發展迅速，生物技術因其具有快速、專一性及靈敏的特性，目前已有多種核酸探針及聚合酵素連鎖反應(PCR)技術被發展應用於鑑定及偵測植物病原菌的研究。然而PCR偵測DNA的靈敏度雖高達50-100 fg，但需要專業技術人才的操作、耗時6.5小時及費用昂貴，所以不易推廣。因此，發展一快速、簡易、高靈敏度且費用低的作物疫病檢測技術，亦有其必要性。本研究擬利用生物奈米技術(bio-nanotechnology)研發具高靈敏度與高光穩定度的百合斑駁病毒螢光奈米診斷探針，並能在短時間內迅速檢測出百合斑駁病毒，供為植物病害檢疫之參考。 7. 艾利希體症檢測技術之開發：艾利希體症是犬及人等之人畜共通傳染病，其病原體主要寄生於單核球，臨床症狀包括體重下降、貧血、淋巴腫大、厭食等症狀。故本試驗將延續先前之研究成果，將犬艾利希體之外膜蛋白Omp30利用生物資訊學進行抗原親水區(hydrophilic region; HR)之預測，再以大腸桿菌表現系統大量表現、純化及定量後，使我們可利用具親水區的Omp30HR或NucA-Omp30HR，作為艾利希體之感染檢測試劑組，並進行艾利希體症病犬之臨床篩檢，以開發具備良好、簡便、靈敏、快速且便宜的檢測試劑，而能有效推動人畜共通傳染病之檢疫防疫計畫。 8. 桃褐腐病菌之診斷偵測：桃褐腐病為對核果類植物最具破壞性之病害之一，此一世界性病害亦曾在台灣被報導過。此病害可由M. fructicola, M. laxa, M. fructigena及M. polystroma感染造成。Monilinia fructicola及M. laxa主要感染核果類植物，而M. fructigena主要感染蘋果及梨，但亦可侵入桃等核果類植物。每種菌各有其被管制之狀態，例如M. fructigena為美國及澳洲兩國禁止輸入之檢疫病菌。為了瞭解國內桃果園所存在及由國外進口水果所攜帶之褐腐病菌種類，必須建立一快速而準確的檢定方法。本研究將收集分離自國內桃果園及進口水果之褐腐病菌，初步菌種鑑定將以培養特性、孢子大小及產孢形態等為依據。以PCR反應為主之分子診斷方法將被建立以了解國內褐腐病菌之種類並可為進出口核果類水果、蘋果及梨等檢測用。本計畫亦將進一步進行國內外桃褐腐病菌株分子層次差異之比對以了解國外菌株與國內菌株之親緣性。 9. 禽類結核病(avian tuberculosis)由鳥型結核桿菌(Mycobacterium avium)所引起之慢性、接觸性、漸進性傳染病，以消化道病變為主，為人畜共通傳染病。本菌對鳥類、豬、貂、兔子、綿羊、鹿、牛等均具敏感性，病禽呈漸進性消瘦、羽毛蓬鬆、下痢等症狀，各臟器均可見多發白色結節，死亡率高。 本計畫擬建立微珠核酸探針技術。輔以病理學檢查、Ziehl-Neelsen氏抗酸染色法、xMAP (Flexible Multi-analyte Profiling)及核酸定序，檢測為禽類結核病。 10. 建立豬瘟疫苗免疫與野外感染區別診斷之技術平台：目前市面上豬瘟疫苗種毒820病毒株，都是由家畜衛生試驗所統一供應，再經動物疫苗廠製作成兔化豬瘟疫苗。台灣至今所收集到的野外豬瘟分離毒共有141株，經核酸部份定序後約可歸類為三種不同的基因型，兔化豬瘟疫苗屬於第一基因型；而現階段田間流行的豬瘟病毒則歸屬於第二基因型。由於接種疫苗及田間感染的病毒株，屬於不同基因型; 田野豬瘟病毒是自然接觸性感染，而兔化豬瘟疫苗是經肌肉注射感染。基於上述兩種不同點，我們認為兔化豬瘟疫苗和田間自然感染病毒，會引起動物產生不同的抗體反應。利用免疫蛋白體學的相關技術，可以將其相對的抗原找出來，抗原找出來後，可利用此抗原製成檢驗試劑，用以區分非感染、野病毒株感染或接種兔化豬瘟疫苗的豬隻，而本年度計畫目標就是企圖找出這樣有差異性的豬瘟抗原。我們預期若實驗順利將可得到非感染、野病毒株感染或接種兔化豬瘟疫苗豬隻的血清抗體可結合的抗原圖譜。經統計分析非感染、野病毒株感染或接種兔化豬瘟疫苗豬隻的血清反應圖譜，找到顯著差異的抗原或未曾發現的抗原。新發現的抗原，可利用MALTI-TOF及LC-MS-MS等技術，定其氨基酸序列，並反推其相對DNA序列，鑑定其身份。之後的延續計畫可利用基因工程法，構築質體在E. coli內大量生產抗原，純化抗原做為檢驗試劑用以鑑別分非感染、野病毒株感染或接種兔化豬瘟疫苗的豬隻。
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