Development of a Taqman RT-PCR assay for the detection and quantification of negatively stranded RNA of human enteroviruses: Evidence for false-priming and improvement by tagged RT-PCR. - Institut Pasteur Access content directly
Journal Articles Journal of Virological Methods Year : 2008

Development of a Taqman RT-PCR assay for the detection and quantification of negatively stranded RNA of human enteroviruses: Evidence for false-priming and improvement by tagged RT-PCR.

Abstract

Human enteroviruses are among the most common viruses infecting humans. These viruses are known to be able to infect a wide range of tissues and are believed to establish persistent infections. Enteroviruses are positive-sense single-stranded RNA viruses whose replication involves the synthesis of negative strand intermediates. Therefore, the specific detection of negatively stranded viral RNA in tissues or cells is a reliable marker of active enteroviral replication. The present report presents the development of a real-time RT-PCR allowing the specific detection and quantification of negatively stranded viral RNA. Since it was known that specific amplification of single-stranded RNA can be made difficult by false-priming events leading to false-positive or overestimated results, the assay was developed by using a tagged RT primer. This tagged RT-PCR was shown to be able to amplify specifically negative RNA of enteroviruses grown in cell cultures by preventing the amplification of cDNAs generated by false-priming.

Domains

Virology

Dates and versions

pasteur-00318587 , version 1 (04-09-2008)

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Cite

Maël Bessaud, Arnaud Autret, Sophie Jegouic, Jean Balanant, Marie-Line Joffret, et al.. Development of a Taqman RT-PCR assay for the detection and quantification of negatively stranded RNA of human enteroviruses: Evidence for false-priming and improvement by tagged RT-PCR.. Journal of Virological Methods, 2008, epub ahead of print. ⟨10.1016/j.jviromet.2008.07.010⟩. ⟨pasteur-00318587⟩

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