Viral load quantitation of SARS-coronavirus RNA using a one-step real-time RT-PCR.
BACKGROUND
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 fast and sensitive diagnostic assays.


RESULTS
A real-time quantitative RT-PCR was designed in the nsp11 region of the replicase 1B domain of the SARS-coronavirus (SARS-CoV) genome. To evaluate 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 quantitation over a range of 10(2) to 10(8) RNA copies per reaction.


CONCLUSIONS
Extrapolated to clinical samples, this novel assay has a detection range of 10(4) to 10(10) copies of viral genome equivalents per millilitre. In comparison to the current de facto cRNA Artus Biotech standard, the in-house cRNA standard gives a 100-fold higher absolute quantity, suggesting a possible underestimation of the viral load when using the Artus Biotech standard.
Keyaerts
Els
E
Laboratory of Clinical and Epidemiological Virology, Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, University Hospital Gasthuisberg, Leuven, Belgium.
Vijgen
Leen
L
Maes
Piet
P
Duson
Griet
G
Neyts
Johan
J
Van Ranst
Marc
M
D000818:Animals; D002522:Chlorocebus aethiops; D017934:Coronavirus / Q000235:genetics / Q000302:isolation & purification*; D028941:Coronavirus 229E, Human / Q000235:genetics / Q000302:isolation & purification; D028962:Coronavirus OC43, Human / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis / Q000821:virology; D014709:Vero Cells
D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase