Quantification of SARS Coronavirus RNA by Real-Time PCR for Rapid Detection of Anti-viral Activity

急性嚴重呼吸道症候群(Severe Acute Respiratory Syndrome, SARS) 由一種新型的冠狀病毒(SARS coronavirus, SARS-CoV)所致，因其高傳染力與高致死率，嚴重威脅人類健康。對於SARS至今仍無有效且特異性高之治療與預防方法，因此疫苗設計和藥物發展刻不容緩。目前評估血清抗體與藥物是否具有中和病毒能力仍以中和試驗為最標準的方法，然而傳統中和試驗往往過於耗時。故本研究擬建立一套即時聚合酶連鎖反應以快速偵測抗SARS-CoV之活性，期以取代傳統的中和試驗。 利用即時聚合酶連鎖反應偵測SARS-CoV RNA，將探針分別設計在SARS-CoV 之RNA依靠RNA聚合酶基因(RNA-dependent RNA Polymerase, RdRP)和核殼基因(Nucleocapsid, NC)。將一定量的SARS-CoV感染Vero E6 細胞，於不同時間點偵測病毒RNA量的變化，發現於感染後八小時即可偵測到病毒RNA量明顯的上升。比較病毒正股RNA與負股RNA之數量，發現兩者之間差距不大；比較偵測不同基因之敏感度，發現RdRP與NC所偵測的結果相差不大。若以恢復期之病人血清及以SARS-CoV合成胜肽免疫之小鼠血清來進行中和試驗，結果顯示若SARS-CoV與具中和能力之血清作用後，則被其感染之細胞中無法以即時聚合酶連鎖反應偵測到病毒之RNA，顯示即時聚合酶連鎖反應可快速偵測血清中抗SARS-CoV之活性。 另外，RNA干擾現象(RNA interference, RNAi)為近年來被認為具有潛力來對抗病毒感染的一個新的研究方向。本研究擬發展以RNAi有效抑制SARS-CoV之生長。實驗中針對SARS-CoV之NC基因設計一段小分子RNA (small interfering RNA, siRNA)，分別以體外轉錄和DNA質體製造。以一定量SARS-CoV病毒感染已轉染siRNA之Vero E6細胞，觀察其細胞病變。初步結果尚未發現有抑制之效果，未來將改進其條件以了解是否可提升抑制之效率，並針對其他基因設計siRNA，以期有效SARS-CoV之生長。Severe Acute Respiratory Syndrome (SARS) has threatened the health of people around the world because of its high transmission and high mortality rate. It is caused by a novel coronavirus, named as SARS coronavirus (SARS-CoV). There are no efficient and specific strategies to treat and prevent SARS up to now. Therefore, it is very urgent and important to develop vaccines and drugs against SARS. Neutralization test is the standard method to evaluate whether or not the sera or drugs have neutralizing activity. However, conventional method is too time-consuming. In this study, a rapid method using real-time polymerase chain reaction (PCR) was established to detect anti-SARS-CoV activity. Real-time PCR was utilized to detect the RNA of SARS-CoV. Probes were designed for the target genes, RNA-dependent RNA polymerase (RdRP) and nucleocapsid (NC) genes. Vero E6 cells were inoculated with SARS-CoV and subsequently the amounts of SARS-CoV RNA were detected at different time points. The amounts of viral RNA could be detected at significant level after 8 hours. Moreover, the amounts detected for plus-sense RNA and minus-sense RNA were similar. Our data also showed that the detection for the NC RNA was not more sensitive than that for the RdRP RNA. Furthermore, the neutralization tests were performed for the sera from patients in the convalescent phase or from mice immunized with synthetic peptides of SARS-CoV. The results showed that no viral RNA could be detected for the SARS-CoV pretreated with sera with neutralizing activity. This study revealed that using real-time PCR could detect the neutralizing activity against SARS-CoV more rapidly. RNA interference (RNAi) is thought to have the potential to fight against virus infection. This study also attempted to develop small interfering RNA (siRNA) with the ability to inhibit the growth of SARS-CoV. First, siRNA targeting the NC gene of SARS-CoV was produced by in-vitro transcription and using DNA vector. After SARS-CoV was inoculated into Vero E6 cells which had been transfected by siRNA, the cytopathic effect was observed. Unfortunately, the preliminary results were not as expected. In the future, the condition of experiments should be optimized to improve the efficiency of inhibition and more target genes for siRNA could be designed to inhibit the growth of SARS-CoV.中文摘要……………………….……………………………………….1 英文摘要……………………….……………………………………….2 序論…………………………………………..………………………….3 實驗材料與方法……………………...…………………………...…12 細胞株和細胞培養…………………….…………………………….12 SARS-CoV 病毒來源……………………………………………….12 SARS病毒RNA之萃取…………………………………………….13 反轉錄反應(reverse transcription, RT)……………..………………..14 聚合酶連鎖反應(Polymerase Chain Reaction, PCR)…………….....14 核酸定序……………………………………………………………..17 質體製備……………………………………………………………..20 即時聚合酶連鎖反應(Real-Time PCR)……………………………..25 SARS-CoV病毒時間點測試 (Time Course)……………………….28 中和試驗(Neutralization Test, NT)…..……………………………...29 RNA干擾現象 (RNA interfering, RNAi)…………………………..29 結果…………………………………………….………………………33 SARS-CoV RNA定量標準品之建立……………………………….33 即時聚合酶連鎖反應定量SARS-CoV RNA系統之建立…………34 SARS-CoV感染細胞後於不同時間點偵測病毒RNA………..……35 SARS-CoV感染細胞後產生正股與負股病毒RNA之比較……….35 以RdRP基因與NC基因偵測病毒RNA之比較………………….36 中和試驗……………………………………………………………..36 RNA干擾試驗……………………………………………………….37 討論…………………………………………………………………….38 圖………………………………………………………………………..46 表………………………………………………………………………..65 參考文獻………………………………………………………………7