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J Virol Methods. 2011 Apr;173(1):1-6. doi: 10.1016/j.jviromet.2010.12.014. Epub 2010 Dec 24.

Strand-specific real-time RT-PCR for distinguishing influenza vRNA, cRNA, and mRNA.

Author information

1
Department of Microbiology and Immunology, University of Tokyo, Tokyo 108-8639, Japan.

Abstract

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 point post-infections. 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 the 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.

PMID:
21185869
PMCID:
PMC3049850
DOI:
10.1016/j.jviromet.2010.12.014
[Indexed for MEDLINE]
Free PMC Article

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