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相關文獻
- Quantitative Analyses of Poliovirus in Seawater and Sewage Using a Competitive Reverse Transcriptase-Polymerase Chain Reaction Method
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- Differentiation of Poliovirus Type 1 Isolates in the 1982 Epidemic from Sabin-1 Strain by Antigenic and Temperature Markers
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- 小兒麻痺病毒
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題 名 | Quantitative Analyses of Poliovirus in Seawater and Sewage Using a Competitive Reverse Transcriptase-Polymerase Chain Reaction Method=利用競爭性反向轉錄-聚合酶連鎖反應來定量分析海水及污水中的小兒麻痺病毒 |
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作 者 | 蔡毓禮; | 書刊名 | 師大生物學報 |
卷 期 | 31:1 1996.06[民85.06] |
頁 次 | 頁19-29 |
分類號 | 415.93152 |
關鍵詞 | 定量性反轉錄-聚合酶連鎖反應; 小兒麻痺病毒; 內定標準; Quantitative RT-PCR; Poliovirus; Internal standard; |
語 文 | 英文(English) |
中文摘要 | 反向轉錄酶及聚合酶連鎖反應(RT-PCR)已經很廣泛的用來測定環境中的樣本是否含有entnc vlruses。利用RT-PCR的優點有高靈敏度的檢測性和快速的報導性。與傳統式的細胞培養相較,RT-PCR並不能使我們得知病毒的數目和感染性有無。在研究過程中,我們發展了一種可定量的RT-PCR方法來測定環境中病毒的數目。我們利用遺傳工程的方法設計了一個RNA內定標準,並將它用於RT-PCR的反應中來測定小兒麻痺病毒的數目。經由連續稀釋過的RNA內定標準模板被混在含有6-FAM標示過的polio3'(下游)端基因引子和polio 5'(上游)端未標示過的基因引子的RT-PCR反應溶液中,DNA定序儀被用來定量經由反應後RT-PCR產品所發出的螢光。在RT-PCR反應中內定標準和小兒麻痺病毒的量同時被放大,而可被用來估定在海水和污水樣本中所含此種病毒的數目。這種利用內定標準和特定基因引子的PCR方法可被引用於定量在環境中某些特定的微生物。 |
英文摘要 | Reverse transcriptase-polymerase chain reaction (RT-PCR) has been used extensively to detect enteric viruses in environmental samples. Advantages of RT-PCR include its high detection sensitivity and rapid turn-around time. However, unlike traditional cell culture, RT-PCR has not provided quantitation and infectivity information. In this study, we have developed a quantitative RT-PCR method which can be used to determine the number of specific viruses in environmental samples. A RNA internal standard for poliovirus RT-PCR was designed and obtained through genetic engineering. Serial dilutions of RNA internal standard templates were amplified with a 6-FAM labeled polio downstream primer and a non labeled polio upstream primer in the RT-PCR. The fluorescent light intensity of labeled RT-PCR products was quantified using an ABI DNA sequencer with GeneScan software. The internal standard was co-amplified with poliovirus in the RT-PCR allowing for enumeration of viruses present in the seawater and sewage samples. This method, using a cloned internal standard and specified primers in the PCR, may be applied to quantify other microorganisms in environmental samples. |
本系統中英文摘要資訊取自各篇刊載內容。