gms | German Medical Science

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.