Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66519
標題: 應用多引子聚合酵素連鎖反應快速診斷豬隻假性狂犬病與小病毒及環狀病毒感染
Multiplex PCR for Rapid Detection of Pseudorabies Virus, Porcine Parvovirus and Porcine Circovirus
作者: 黃千衿
鍾明華
簡茂盛
張天傑
關鍵字: 畜牧獸醫類
應用研究
摘要: 計畫目標: 開發多引子聚合酵素連鎖反應之快速診斷方法, 可同時檢測豬隻是否遭受假性狂犬病毒、小病毒或環狀病毒感染並完成此三種病毒之PCR診斷技術的標準流程作業手冊.架構( 重要工作項目 ): 1.引子( primer )之設計根據假性狂犬病毒( PRV )之gB、gD、gE、gX、IE基因與豬小病毒( PPV )之VP2、NS-1基因及豬環狀病毒( PCV )之PVC-1、PVC-2等基因序列分別設計多組之PCR引子.這些寡核甘酸引子經合成及純化後, 分別進行PCR反應以測定各引子對之專一性與敏感性.2.病毒核酸的純化PRV、PPV與PCV等病毒分別進行病毒培養並自各病毒感染的組織培養細胞中純化病毒核酸以作為陽性對照組.而感染豬隻之臨床組織樣本( 包括脾臟、肺臟、肝臟、淋巴結、扁桃腺等 )先以PBS製備成20%懸浮液後, 一方面進行病毒分離, 另一方面進行病毒核酸的萃取純化.3.多引子聚合酵素連鎖反應最佳反應條件之測定各病毒之引子對皆先分別測試其特異性及敏感性後即進行多引子PCR中各項反應條件之測試, 以選取最適當之反應條件.反應條件的優化( optimization )主要根據特異性及敏感性而調整反應溫度、循環次數及各反應物之濃度, 並設定多引子聚合酵素連鎖反應結果之分析程序及判定標準.4.動物試驗大量收集各豬場之豬隻臨床樣本( 包括血液、扁桃腺拭子、病豬組織等 ), 應用多引子PCR反應來進行假性狂犬病、豬小病毒及豬環狀病毒感染的篩選, 並與血清學調查的結果互相比較分析.預期效益: 所建立之多引子聚合酵素連鎖反應方法可以快速區別診斷出豬隻是否遭受假性狂犬病毒、小病毒或環狀病毒的感染, 有助於豬病的預防與清除.
Pseudorabies virus ( PRV )can cause severe disease-Aujeszky's disease ( pseudorabies )and lead to latent infection in swine.Porcine parvovirus ( PPV )is the major cause of SMEDI ( stillbirth, mummification, embryonic death, infertility )syndrome.Porcine circovirus ( PCV )has been considered as a persistent contaminant of the continuous porcine kidney cell line, and newly identified strain is associated with postweaning multisystemic wasting syndrome ( PMWS )in growing pigs.Viruses isolation studies and serological surveys have demonstrated that the PRV, PPV, and PCV infections are widespread worldwide.The purpose of this project is to develop a diagnostic method by multiplex PCR analysis for the rapid detection and differentiation of PRV, PPV, and PCV in clinical specimens.For setting up PCR reactions, the PCR primers should be carefully designed.Several primer pairs specific to PRV, PPV and PCV are synthesized according to the published gene sequences, and the specificity and sensitivity of each primer is also determined.Tissue samples from infected pigs are prepared in 20%homogenates and the viral DNA is extracted from tissue homogenates for multiplex PCR testing of viruses.The results obtained from this project can provide important information for establishing the standard operation procedure ( SOP )of PCR diagnosis to pseudorabies virus, porcine parvovirus and porcine circovirus respectively.The multiplex PCR method will offer a rapid detection and differentiation of these viruses that will assist the control and eradication of important swine diseases.
URI: http://hdl.handle.net/11455/66519
其他識別: 90農科-6.2.2-檢-B3(3)
文章連結: http://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=1041391&plan_no=90%E8%BE%B2%E7%A7%91-6.2.2-%E6%AA%A2-B3%283%29&plan_year=90&projkey=PG9309-1826&target=plan&highStr=*&check=0&pnchDesc=%E6%87%89%E7%94%A8%E5%A4%9A%E5%BC%95%E5%AD%90%E8%81%9A%E5%90%88%E9%85%B5%E7%B4%A0%E9%80%A3%E9%8E%96%E5%8F%8D%E6%87%89%E5%BF%AB%E9%80%9F%E8%A8%BA%E6%96%B7%E8%B1%AC%E9%9A%BB%E5%81%87%E6%80%A7%E7%8B%82%E7%8A%AC%E7%97%85%E8%88%87%E5%B0%8F%E7%97%85%E6%AF%92%E5%8F%8A%E7%92%B0%E7%8B%80%E7%97%85%E6%AF%92%E6%84%9F%E6%9F%93
Appears in Collections:微生物暨公共衛生學研究所

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