Quantitative Analyses of Poliovirus in Seawater and Sewage Using a Competitive Reverse Transcriptase-Polymerase Chain Reaction Method

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entric viruses。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內定標準模板被混合在含有6-FAM標示過的polio 3（下游）端基因因子和polio 5（上游）端未標示過的基因因子的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，而可用來估定在海水和汙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。

關鍵字

定量性反轉錄－聚合酶連鎖反應；小兒麻痺病毒；內定標準

並列摘要

Reverse trunscnptasc-polymcrasc 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.

並列關鍵字

Quantiative RT-PCR ； poliovirus ； internal standard

國際替代計量

Quantitative Analyses of Poliovirus in Seawater and Sewage Using a Competitive Reverse Transcriptase-Polymerase Chain Reaction Method

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