gms | German Medical Science

International Conference on SARS - one year after the (first) outbreak

08. - 11.05.2004, Lübeck

A real-time quantitative RT-PCR for absolute quantification of SARS coronavirus RNA

Poster

  • corresponding author presenting/speaker Leen Vijgen - Rega Institute for Medical Research, University of Leuven, Leuven, Belgium.
  • Els Keyaerts - Rega Institute for Medical Research, University of Leuven, Leuven, Belgium.
  • Griet Duson - Rega Institute for Medical Research, University of Leuven, Leuven, Belgium.
  • Johan Neyts - Rega Institute for Medical Research, University of Leuven, Leuven, Belgium.
  • Marc van Ranst - Rega Institute for Medical Research, University of Leuven, Leuven, Belgium.

International Conference on SARS - one year after the (first) outbreak. Lübeck, 08.-11.05.2004. Düsseldorf, Köln: German Medical Science; 2004. Doc04sarsP8.02

Die elektronische Version dieses Artikels ist vollständig und ist verfügbar unter: http://www.egms.de/de/meetings/sars2004/04sars119.shtml

Veröffentlicht: 26. Mai 2004

© 2004 Vijgen et al.
Dieser Artikel ist ein Open Access-Artikel und steht unter den Creative Commons Lizenzbedingungen (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.de). Er darf vervielf&aauml;ltigt, verbreitet und &oauml;ffentlich zug&aauml;nglich gemacht werden, vorausgesetzt dass Autor und Quelle genannt werden.


Gliederung

Text

Severe acute respiratory syndrome (SARS) is an emerging infectious disease that first occurred in humans in the People's Republic of China in November 2002 and has subsequently spread worldwide. A novel virus belonging to the Coronaviridae family has been identified as the cause of this pulmonary disease. The severity of the disease combined with its rapid spread requires the development of rapid and sensitive diagnostic tests. A real-time quantitative RT-PCR (Taqman®) was designed in the putative nsp11 region in the replicase 1B domain of the SARS-associated coronavirus (SARS-CoV) genome. The following oligonucleotides were used for the amplification of a 68 bp fragment: forward primer (SARS-FP: 5'-CACCCGCGAAGAA GCTATTC-3'), MGB probe (SARS-TP: FAM 5'-TGCGTGGATTGGCTT-3' NFQ-MGB) and reverse primer (SARS-RP: 5'-TTGCATGACAGCCCTCTACATC-3'. To determine the sensitivity of this quantitative RT-PCR, cRNA standards were constructed by in vitro transcription of SARS-CoV Frankfurt 1 RNA using T7 RNA polymerase, followed by real-time RT-PCR. The assay allowed quantification over a range from 102 to 108 RNA copies per reaction (5 μl RNA extract). Extrapolated to clinical samples, this corresponds to a detection range of 104 to 1010 copies of viral genome equivalents per ml. In this study we present a one-step real-time quantitative RT-PCR assay as a highly sensitive method for the rapid diagnosis of SARS-CoV. In comparison with the current de facto cRNA standard of Artus Biotech, our standard gives an 100 fold higher absolute quantity. The method here presented allows rapid identification of new SARS cases which is of major importance to control a possible new outbreak.