應用反轉錄聚合酶連鎖反應鑑定暨區分新城雞病病毒疫苗株及野外株

為了鑑定區分新城雞病病毒疫苗株與野外強毒株，我們利用反轉錄聚合酶鏈反應技術，建立快速區分新城雞病病毒的方法。首先，我們選擇了一組對新城雞病病毒具專一性的引子進行RT-PCR，當待測物含有病毒時，我們可增幅出一段426 base pairs (bp)產物，再將此產物分別以限制酶HinP1 Ⅰ與HaeⅢ作用，台灣株只會被HinP1 Ⅰ作用，而疫苗株則只能被Hae Ⅲ作用，藉此可區分新城雞病病毒疫苗株與野外株。另一方面，我們又設計了三段對新城雞病病毒具專一性的引子，進行多引子反轉錄聚合酶鏈反應，若病材含有新城雞病病毒野外株時，就可增幅一段724 bp產物，反之，若有疫苗株存在時，則會增幅出349 bp的產物，因此也可區分新城雞病病毒疲苗株與野外株。由本實驗的結果證明，RT-PCR配合限制酶切割，或直接進行多引子RT-PCR反應，皆可應用於新城雞病病毒之快速區別診斷。這是第一篇應用多引子RT-PCR來診斷新城雞病病毒之報告。

關鍵字

新城雞病；反轉錄聚合酶鏈反應

並列摘要

We developed a rapid and simple procedure, based on the reverse transcription-polymerase chain reaction (RT-PCR), to identify and differentiate Taiwanese field isolates and vaccine strains of Newcastle disease virus (NDV). A 426 base-pairs (bp) fragment was amplified from samples containing NDV by RT-PCR, and then digested by restriction endonucleases HinP1 Ⅰand Hae Ⅲ. The fragment amplified from Taiwanese isolates could be digested by Hin P1 Ⅰbut not by Hae Ⅲ. In contrast, the fragment amplified from vaccine strains could be digested by Hae Ⅲ but not by HinP1 T. Therefore, RT-PCR combined with restriction enzyme digestion provides a convenient procedure for discriminating Taiwanese field isolates from vaccine strains of NDV. In addition, we developed a multiple-primers RT-PCR procedure for the identification and differentiation of NDV. In this procedure, three primers were included in the RT-PCR, 50 that a 724 bp fragment of HN gene was amplified from samples containing Taiwanese isolates, while a 349 bp fragment was amplified from samples containing vaccine strain s. In this way, we could differentiate field isolates of NDV from vaccine strains by a simple RTPCR assay. Results of this study demonstrate that RT-PCR provides a simple and rapid method for the detection and differential diagnosis of NDV. This is the first report on using multiple-primers RT-PCR in the diagnosis of NDV.

並列關鍵字

Newcastle disease ； Reverse transcription-polymerase chain reaction

國際替代計量

應用反轉錄聚合酶連鎖反應鑑定暨區分新城雞病病毒疫苗株及野外株

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