Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52661
標題: Development and Application of Diagnostic Techniques for Animal Diseases
動物疫病診斷鑑定技術之研發與應用
作者: 張天傑
楊平政
劉振軒
黃千衿
王孟亮
費昌勇
陳石柱
鄭明珠
曾浩洋
張伯俊
邱賢松
林永昌
賴秀穗
龐飛
蔡信雄
劉宏仁
胡仲祺
關鍵字: 畜牧獸醫類
應用研究
摘要: 1. The foot and mouth disease (FMD) eradication program has been an important policy in Taiwan, and it leads to be recognized as a FMD free country. At present, commercialized antibody detection kits mainly use the method of enzyme-linked immunosorbent assay (ELISA). In this project, we are planning to develop an antibody detection kit using immunodot method. The antigen used will be expressed protein of VP1, VP2, and 3AB genes of FMD virus. This newly developed antibody detection kit will be able to be used in the field without using expansive equipment. This development should be very helpful to the execution of the FMD eradication campaign in the future. 2. HPAI of the H5 subtype has imposed a great potential threat to both the poultry industry and humans in Taiwan. To effectively monitor and manage the disease, we have previously developed a rapid virus detection kit based on the monoclonal antibodies against the HA protein of avian influenza virus H5 subtype for the on-site operations of the front-line workers. To further test the sensitivity and specificity of the rapid detection kit with clinical samples, we will continue the collaboration with Animal Health Research Institute to perform the virus challenge assays with H5N2 (or H5N1) and H6 subtypes of AIV under Bio-Safety Level 3 environments. Scheduled works includes the clinical evaluation for the sensitivity, specificity of the rapid virus detection kits, as well as training and extension for the use of the kit. It is expected that the rapid virus detection kit for avian influenza virus H5 subtype will serve as a tool for the efficient, rapid and initial on-site screening of the diseases, and facilitate the effective control of the viruses to ensure the sustainable poultry industry in Taiwan. 3. Enterotoxigenic E. coli (ETEC), Salmonella Typhimurium, S. Choleraesuis, Clostridium perfringens etc., are major pathogens for porcine diarrhea cases. On the other hand, Staphylococcus aureus, some Streptococcus spp. and Corynebacterium bovis may cause mastitis for bovine. In comparison with conventional PCR, Real-time PCR has recently become a popular method. It does not require the step of gel electrophoresis, is time saving, more sensitive and causes non-cross reaction. In this study, by use of SYBR Green or TaqMan Probe, we shall design Real-time PCR method for the specific detection of Salmonella Typhimurium, S. Choleraesuis, ETEC and Clostridium perfringens. Meanwhile, Real-time PCR quantation for these pathogenic bacteria will be tried since pathogenicity of bacteria is highly correlated with its cell number. Finally, we shall also try the possibility to develop a multiplex Real-time PCR system for the simultaneous detection of these diarrheagenic bacteria. Through this project, we expect to establish a rapid Real-Time PCR method which allows us to trace the cause of diarrhea and infections for porcine and thus prevents the spreading of infection. Since S. Typhimurium, S. Choleraesuis, ETEC, and C. perfringens are common pathogens for human and porcine, this method can be usd to trace the contamination sources for both human and porcine infections. Finally, we shall also try to develop a multiplex Real-Time PCR system and use it for the simultaneous detection of these pathogenic bacteria. Also, patent application and technique transfer to bioindustry will be attempted. 4. Hepatitis E is a non-A, non-B hepatitis and is transmitted by the fecal-oral route. Contamination of drinking water has become the main reason to cause outbreaks of hepatitis E in several developing countries. It is an important disease with public health concern. The mortality rate of hepatitis E is generally low, about 1% only; however, it can be more serious in
一、口蹄疫抗體快速檢測試劑之研發:口蹄疫的撲滅計畫已被列為我國的重大防疫政策,而目前實驗室中對於口蹄疫的診斷方法有螢光抗體檢測法、病毒分離、RT-PCR及免疫吸附法(ELISA)等方法,以上檢測方法所需時間長,且需要特殊儀器,無法於現場使用,因此需開發一方便快速的檢驗方法,以便在現場快速檢測並立即摘除帶原者。本實驗將針對口蹄疫病毒的VP1、VP2及3AB基因,來進行原核系統的表現與純化,以開發出一種較現有檢測方法更具有經濟效益及特異性、敏感性、簡便而快速的口蹄疫抗體檢測方法以提供臨床檢測之用。本計畫將與國內知名且具高度研發生產及行銷能力佳之生物技術公司進行產學合作,利用他們的免疫墨渍檢測法平台開發出快速現場檢測口蹄疫抗體的診斷試劑套組,並期待於未來予以商品化,未來將有助於協助監測口蹄疫疫苗注射狀況,及進行現場口蹄疫病毒感染豬隻檢測與清除。 二、流感病毒H5亞型快速檢測試劑套組之實測與應用:H5亞型之高病原性家禽流行性感冒(HPAI)對台灣養禽業具極大之潛在危險,並有人畜共通疾病之隱憂。為求有效地監控此病毒,本計劃上年度已初步完成禽流感病毒H5亞型病毒快速檢測試劑套組。本年度由中興大學與家畜衛生試驗所合作,繼續以H5N2 (或H5N1)與H6N1亞型禽流感病毒進行攻毒試驗。預期將可利用禽流感病毒時體樣本完成禽流感病毒快速檢測套組之實體樣本測試與評估,並進行人員訓練與推廣工作,未來可供第一線工作人員作為現場快速病毒篩檢之基本工具,防堵病毒入侵與擴散,確保我國養禽產業之永續經營。 三、開發多套式即時聚合酶鏈鎖反應快速檢測豬下痢病原菌及牛乳房炎病原菌之技術及其應用:豬下痢及腸炎之病原菌,包括腸毒型大腸桿菌(ETEC)、沙門氏菌(如S. Typhimurium、S. Choleraesuis)、腸道螺旋體、Clostridium perfringens…等,另一方面,造成牛乳房炎的病原菌,則包括葡萄球菌、鏈球菌、棒狀桿菌、大腸菌…等。與傳統PCR比較,Real-Time PCR 不需電泳操作且靈敏度提高,受檢測菌數不必增殖,故能大大減少縮短檢測時間,並避免偽陽性的發生。因此本研究擬利用Real-Time PCR配合SYBR Green染劑或TaqMan probe方法,設計沙門氏菌屬如S. Typhimurium、S. Choleraesuis及ETEC、產氣莢膜桿菌專一性引子組及專一性TaqMan探針,用以快速鑑定禽畜類重要之感染性病原菌,由於致病性與菌量亦有相關性;因此發展同時檢測多種病原菌並定量之快速方法於防治感染是必要的。本計畫擬分年開發多套式Real-Time PCR,針對上述細菌中S. Typhimurium, S. Choleraesuis、ETEC(LT及ST)及Clostridium perfringens等設計專一性引子組及TaqMan探針,以Real-Time PCR檢測定量豬隻感染之重要病原菌。本研究預計可建立一套快速檢測豬隻下痢病原菌的即時PCR套組(Real Time PCR),了解豬隻下痢之病因,達到傳染性胃腸炎之快速防制、避免擴散等目的。S. Typhimurium、S. Choleraesuis、ETEC(含heat-liable toxin, LT, 及heat stable toxin, ST)及產氣莢膜桿菌為人畜共通病原菌,本方法亦可應用於人類、豬隻感染上述病原菌之病因,對相關傳染途徑予以控制。並可設計一套多重檢測之 Real-Time PCR套組,用於多種細菌之同時檢測。相關PCR套組可申請專利移轉生物技術產業使用。 四、人畜共通傳染病原: 豬E型肝炎病毒快速檢測技術之開發:E型肝炎是經口糞感染的非A及非B型肝炎,在開發中國家因水源遭受污染常有爆發性流行,為一重要的公共衛生疾病。E型肝炎感染者一般死亡率約在1%左右,但在孕婦,尤其第三期妊娠感染者,死亡率可達20%。E型肝炎病毒在豬隻身上亦被發現,在日本曾有因直接生食野鹿肉或野豬肝臟而導致發病死亡的病例;此病原在豬隻飼養、屠宰及獸醫師等相關人員有明顯高血清抗體陽性率。台灣豬E型肝炎病毒感染相關情形雖有部分報告,但相關的研究及疫情掌握仍有不足,為因應未來此一人畜共通傳染病及畜產品產製環境之人畜安全檢測需要,本計劃擬建立及開發豬E型肝炎病毒之快速診斷技術及試劑。 五、豬流感病毒單源抗體製備、特性分析與診斷套組開發:臺灣豬群中流行的A型流行性感冒病毒,則除了古典豬型H1N1亞型及似人型H3N2亞型之外,近期也偵測出H1N2亞型及H3N1亞型等二種新興病毒變異株。鑑於現有檢驗診斷技術方法已不足以有效偵測新型或
URI: http://hdl.handle.net/11455/52661
其他識別: 95農科-13.3.1-檢-B3
文章連結: http://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=1271608&plan_no=95%E8%BE%B2%E7%A7%91-13.3.1-%E6%AA%A2-B3&plan_year=95&projkey=PG9507-3504&target=plan&highStr=*&check=0&pnchDesc=%E5%8B%95%E7%89%A9%E7%96%AB%E7%97%85%E8%A8%BA%E6%96%B7%E9%91%91%E5%AE%9A%E6%8A%80%E8%A1%93%E4%B9%8B%E7%A0%94%E7%99%BC%E8%88%87%E6%87%89%E7%94%A8
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