Nucleic Acid Detection of Rabies Virus RNA

為開發狂犬病病毒核酸偵測之能力以備不時之需，本實驗嚐試利用狂犬病疫苗毒建立以逆轉錄—聚合酶鏈反應（reverse transcriptase-polymerase chain reaction,　RT-PCR）與點雜交反應（dot hybridization）偵測狂犬病病毒之方法。首先針對狂犬病病毒的核蛋白（nucleoprotein, N）與基質蛋白（matrix protein, M）基因設計引子，利用RT-PCR自狂犬病疫苗中放大獲得一段長度為422 bp的DNA片段。此片段以核酸定序加以分析，確認為狂犬病病毒M基因之一部份。進一步以質體將此片段加以選殖，並分別利用T7與SP6 RNA聚合酶進行試管內轉錄反應（in vitro transcription），合成正向與負向之digoxigenin（DIG）標定RNA探針。在點雜交反應中以重組質體DNA與狂犬病疫苗中萃取出之總RNA作為雜交目標分子，結果發現不論正向與反向之RNA探針均可產生清晰的陽性訊號；其強度則隨雜交目標分子之不同而出現差異。實驗結果並顯示，僅需0.47 ng的微量疫苗總RNA萃取物，即可令正向與反向之探針產生可辨識的陽性訊號。此結果顯示本實驗中所發展出的RT-PCR與雜交技術能夠快速而敏感的偵測狂犬病病毒核酸，未來將能成為輔助傳統狂犬病診斷方法之工具。

關鍵字

診斷；點雜交反應； M基因；狂犬病；逆轉錄—聚合酶鏈反應

並列摘要

In order to establish sensitive diagnostic assays for the rapid detection of rabies virus, tests using reverse transcriptase-polymerase chain reaction (RT-PCR) and non-radioactive dot hybridization were developed. Primers based on the conserved sequences of nucleoprotein (N) and matrix protein (M) genes of rabies virus genome were designed for the RT-PCR test. A 422 bp DNA fragment was amplified from a vaccine virus strain and subsequently confirmed to be a part of the M gene of rabies virus by sequencing. This gene fragment was cloned and further applied in the probe preparation for the establishment of a non-radioactive dot hybridization test. Digoxigenin-(DIG-) labeled sense and antisense RNA probes were generated using T7 and SP6 RNA polymerase by an in vitro transcription system. Strong positive hybridization signals were obtained by both sense and antisense probes when the nylon membranes were spotted with the total RNA extract from rabies vaccine. The sense RNA probe could detect with as little as 0.47 ng of the total RNA of rabies vaccine preparation. The results indicate that the M gene based RTPCR and the DIG-Iabeled hybridization tests developed in this study can be applied as sensitive tools for the diagnosis of rabies infection.

並列關鍵字

Diagnosis ； Dot hybridization ； M gene ； Rabies ； RT-PCR

國際替代計量

Nucleic Acid Detection of Rabies Virus RNA

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