Strand-specific real-time RT-PCR for distinguishing influenza vRNA, cRNA, and mRNA.
Strand-specific real-time RT-PCR for distinguishing influenza vRNA, cRNA, and mRNA.
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DOI：
10.1016/j.jviromet.2010.12.014
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发表时间：
2011-04
影响因子：
3.1
通讯作者：
Kawaoka, Yoshihiro
中科院分区：
文献类型：
--
作者：
Kawakami, Eiryo;Watanabe, Tokiko;Fujii, Ken;Goto, Hideo;Watanabe, Shinji;Noda, Takeshi;Kawaoka, Yoshihiro
关键词：
实时RT-PCR用于定量单个流感病毒RNA。然而，使用链特异性引物的常规实时RT-PCR已显示出不仅产生预期的链特异性产物，而且产生大量的非链特异性产物，表明缺乏特异性。因此，本研究建立了一种新的链特异性实时RT-PCR方法，分别定量流感病毒的三种类型的RNA（vRNA，cRNA和mRNA）。该方法基于使用标记引物在5端添加标签序列的逆转录和热启动方法。使用标签部分作为正向引物和片段特异性反向引物的实时PCR确保了用于定量三种类型的RNA的特异性。使用这种方法，特异性靶RNA的检测水平比其他类型的RNA高100 - 100，000倍。该方法还用于评价感染后不同时间点用甲型流感病毒感染的MDCK细胞中片段5和6的vRNA、cRNA和mRNA水平。cRNA水平比vRNA和mRNA水平低1/10 ~ 1/100。此外，观察到vRNA、cRNA和mRNA合成的不同动力学;在整个感染过程中，vRNA的拷贝数逐渐增加，cRNA增加然后趋于稳定，而mRNA增加然后下降。因此，这种新方法提供了了解流感病毒生命周期的关键数据，包括转录，复制和基因组掺入病毒体。
Real-time RT-PCR is used to quantify individual influenza viral RNAs. However, conventional real-time RT-PCR, using strand-specific primers, has been shown to produce not only the anticipated strand-specific products, but also substantial amounts of non-strand-specific products, indicating lack of specificity. Therefore, in this study, a novel strand-specific real-time RT-PCR method was established to quantify the three types of influenza viral RNA (vRNA, cRNA, and mRNA) separately. This method is based on reverse transcription using tagged primers to add a tag sequence at the 5 end and the hot-start method. Real-time PCR using the tag portion as the forward primer and a segment-specific reverse primer ensured the specificity for quantifying the three types of RNA. Using this method, specific target RNA was detected at 100 – 100,000 folds higher level than other types of RNA. This method was also used to evaluate the vRNA, cRNA, and mRNA levels of segments 5 and 6 in MDCK cells infected with influenza A virus at different time points post-infection. The cRNA level was 1/10 to 1/100 lower than that of the vRNA and mRNA. Moreover, different dynamics of vRNA, cRNA, and mRNA synthesis were observed; the copy number of the vRNA gradually increased throughout infection, the cRNA increased and then plateaued, while the mRNA increased and then decreased. This novel method thus provides data critical for understanding the influenza virus life cycle, including transcription, replication, and genome incorporation into virions.
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影响因子：
2.7
作者：
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通讯作者：
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通讯作者：
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