Development and Application of a Multiplex Reverse Transcription-Polymerase Chain Reaction for Avian Viral Respiratory Agents

發展及應用可偵測家禽病毒性呼吸道病原之多引子反轉錄聚合酶鏈鎖反應

黃元品(Yuan-Pin Huang) ； 王金和(Ching-Ho Wang)

《臺灣獸醫學雜誌》 34卷1期 (2008/03) Pp. 8-18

https://doi.org/10.7009/TVJ.200803.0008

摘要

呼吸道感染對於家禽產業的影響很大，而使用單引子反轉錄聚合酶鏈鎖反應來作臨床診斷需要進行數次的檢驗。為了節省檢驗所需要的時間，我們發展出可以在同一個反應中偵測出數種家禽病毒性呼吸道病原的多引子反轉錄聚合酶鏈鎖反應。我們使用針對家禽流行性感冒病毒(AIV)、傳染性支氣管炎病毒(IBV)、傳染性喉頭氣管炎病毒(ILTV)、新城病病毒(NDV)的特異性引子來進行測試。結果顯示多引子或單引子的反轉錄聚合酶鏈鎖反應皆有相同的預期產物。此多引子反轉錄聚合酶鏈鎖反應對ILTV、IBV、NDV、AIV的敏感度分別為可偵測到3.5ng、0.14ng、0.4ng、0.36ng的病毒核酸。在22個病毒分離陽性的檢體中，有77%的比例可以從乳劑中直接測得病毒核酸；而64個病毒分離陰性的檢體中，有91%的比例是乳劑中病毒核酸檢測陰性。因此，此種多引子反轉錄聚合酶鏈鎖反應可以作為一種在臨床病材中對於家禽病毒性呼吸道病原的快速檢驗及區分方法。

關鍵字

家禽流行性感冒病毒 ； 傳染性支氣管炎病毒 ； 傳染性喉頭氣管炎病毒 ； 多引子 ； 新城病病毒 ； 反轉錄聚合酶鏈銷反應

並列摘要

Respiratory tract infections are critical in the poultry industry. Separated reverse transcription-polymerase chain reaction (RT-PCR) assays for different avian diseases require several reactions. A multiplex RT-PCR assay that detects avian viral respiratory pathogens in the same reaction requires less time for clinical practice. Four sets of specific oligonucleotide primers for infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and avian influenza virus (AIV) were used in this study. Both multiplex RT-PCR and single RT-PCR resulted in expected products. The sensitivity limits of this multiplex RT-PCR were 3.5ng, 0.14ng, 0.4ng, and 0.36ng for ILTV, IBV, NDV, and AIV, respectively. Among the tested samples, seventy-seven percent of the 22 virus isolation-positive samples showed positive by genome detection of homogenates. In addition, ninety-one percent of the 64 virus isolation-negative samples showed negative by genome detection of homogenates. Thus, the multiplex RT-PCR was able to detect pathogens in clinical samples for rapid identification and differential diagnosis of poultry viral respiratory infections.

並列關鍵字

AIV ； IBV ； ILTV ； multiplex ； NDV ； RT-PCR