2015-06-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/697853摘要：犬呼吸道感染常見於犬隻，其感染通常為多病原的，許多病毒及細菌已被報導為可能的病原菌，不同的呼吸道病原於呼吸道感染初期時所造成的臨床症狀相似而造成鑑別診斷上的困難。基因晶片可同時進行多個病原檢測。為了可以特異的敏感的進行犬呼吸道病原的偵測及鑑別，本研究擬建立了一個針對犬隻呼吸道感染的診斷基因晶片，偵測病原包括犬瘟熱病毒 (Canine distemper virus)、犬流感病毒 (Canine influenzavirus)、犬疱疹病毒 (Canine herpesvirus)、犬黴漿菌 (Mycoplasma cynos) 及支氣管敗血性博得氏菌 (Bordetella bronchiseptica)。此基因晶片除了偵測上述五種病原外，亦同時可進行犬瘟熱病疫苗毒株及野外毒株的區分。針對五種病原設計引子對建立多引子聚合酶鏈鎖反應，將多引子聚合酶鏈鎖反應的產物，基因晶片上依據各標定病原的特定序列設計的寡核苷酸探針進行雜交反應。以肉眼進行結果判讀，可見各病原的寡核苷探針皆能產生清晰且具特異性的雜交訊號。進一步利用基因轉殖的技術，分別計算DNA與RNA病原分子的複製數（copy number），測試基因晶片的檢測極限。最後應用施行於臨床樣本的檢測，併進行晶片平台的探針或實驗條件的修正。 本研究所建立之犬呼吸道診斷基因晶片將大有助於診斷技術的改良精進。 <br> Abstract: Canine respiratory infection is common in dogs worldwide. The infection appears to be multifactorial and a number of viruses and bacteria have been reported as potential pathogens. The clinical signs of early infection caused by different respiratory pathogens are similar and difficult in differentiation. Gene chip could analyze multiple pathogens at a time. To detect and differentiate canine respiratory pathogens with a higher sensitivity and specificity approach, a diagnostic DNA gene chip targeting at canine distemper virus, canine influenza virus, Mycoplasma cynos and Bordetella bronchiseptica was attempted. Besides these five specific pathogens, this assay could differentiate canine distemper virus vaccine strains from field strains. Multiplex RT-PCR using a set of primers for targeting all pathogens was established. The PCR products were then hybridized with the gene chip oligonucleotide probes based on the specific nucleotide sequences. Clear hybridization signals that could detect and differentiate the targeting pathogens were successfully developed and were read by naked eye. Pathogen DNA or RNA molecule copy numbers were calculated using gene cloning methods to test the chip detection limit. Finally, the developed chip system will be applied to the clinical samples and further modification of probes or experimental conditions will be performed. The study results will greatly assist in the diagnosis of canine respiratory diseases.基因晶片犬呼吸道感染犬瘟熱病毒犬流感病毒犬疱疹病毒支氣管敗血性博得氏菌犬黴漿菌多引子聚合酶鏈鎖反應Gene chipcanine respiratory infectioncanine distemper viruscanine influenza viruscanine herpesvirusMycoplasma cynosBordetella bronchisepticamultiplex PCR犬呼吸道病原偵測及分型基因晶片之研發(1/2)