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Quantification of in vitro retroviral replication using a one-tube real-time RT-PCR system incorporating direct RNA preparation.


Bisset, L R; Bosbach, S; Tomasik, Z; Lutz, H; Schüpbach, J; Böni, J (2001). Quantification of in vitro retroviral replication using a one-tube real-time RT-PCR system incorporating direct RNA preparation. Journal of Virological Methods, 91(2):149-155.

Abstract

The methodological and logistic benefits gained from assessing in vitro antiretroviral replication using one-tube real-time RT-PCR procedures are currently diminished by a continuing need for prior RNA isolation. We now report a simple and inexpensive modification of a commercially available one-tube RT-PCR assay, consisting of detergent-based virus lysis in the presence of a ribonuclease inhibitor, which can be used to directly quantify retroviral RNA levels in culture supernatant. This approach circumvents the potential loss of RNA inherent to RNA-isolation procedures based on prior extraction and demonstrates a dynamic range of at least 4 logs. Using in vitro culture systems incorporating either HIV-1 or FIV, we show that this ability to isolate retroviral RNA directly during the RT-PCR process can provide an equivalent alternative to one of the more time and resource-consuming steps in quantifying in vitro retroviral RNA levels.

The methodological and logistic benefits gained from assessing in vitro antiretroviral replication using one-tube real-time RT-PCR procedures are currently diminished by a continuing need for prior RNA isolation. We now report a simple and inexpensive modification of a commercially available one-tube RT-PCR assay, consisting of detergent-based virus lysis in the presence of a ribonuclease inhibitor, which can be used to directly quantify retroviral RNA levels in culture supernatant. This approach circumvents the potential loss of RNA inherent to RNA-isolation procedures based on prior extraction and demonstrates a dynamic range of at least 4 logs. Using in vitro culture systems incorporating either HIV-1 or FIV, we show that this ability to isolate retroviral RNA directly during the RT-PCR process can provide an equivalent alternative to one of the more time and resource-consuming steps in quantifying in vitro retroviral RNA levels.

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15 citations in Web of Science®
18 citations in Scopus®
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Additional indexing

Item Type:Journal Article, refereed
Communities & Collections:04 Faculty of Medicine > Institute of Medical Virology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:1 February 2001
Deposited On:11 Feb 2008 12:28
Last Modified:05 Apr 2016 12:22
Publisher:Elsevier
ISSN:0166-0934
Publisher DOI:10.1016/S0166-0934(00)00259-7
PubMed ID:11164496

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