Abstract:
:Using a multiplexed LNA-based Taqman assay, RT-digital PCR (RT-dPCR) was performed in a prefabricated microfluidic device that monitored absolute viral load in native and immortalized cell lines, overall precision of detection, and the absolute detection limit of an occult RNA virus GB Virus Type C (GBV-C). RT-dPCR had on average a 10% lower overall coefficient of variation (CV, a measurement of precision) for viral load testing than RT-qPCR and had a higher overall detection limit, able to quantify as low as three 5'-UTR molecules of GBV-C genome. Two commercial high-yield in vitro transcription kits (T7 Ribomax Express by Promega and Ampliscribe T7 Flash by Epicentre) were compared to amplify GBV-C RNA genome with T7-mediated amplification. The Ampliscribe T7 Flash outperformed the T7 Ribomax Express in yield of full-length GBV-C RNA genome. THP-1 cells (a model of monocytic derived cells) were transfected with GBV-C, yielding infectious virions that replicated over a 120h time course and could be infected directly. This study provides the first evidence of GBV-C replication in monocytic derived clonal cells. Thus far, it is the only study using a microfluidic device that measures directly viral load of mammalian RNA virus in a digital format without need for a standard curve.
journal_name
J Virol Methodsjournal_title
Journal of virological methodsauthors
White RA 3rd,Quake SR,Curr Kdoi
10.1016/j.jviromet.2011.09.017subject
Has Abstractpub_date
2012-01-01 00:00:00pages
45-50issue
1eissn
0166-0934issn
1879-0984pii
S0166-0934(11)00392-2journal_volume
179pub_type
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