Viral load quantitation of SARS-coronavirus RNA using a one-step real-time RT-PCR

Abstract

Introduction: 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.

Figure 1

Ten-fold serial dilutions ranging from 10⁸ to 10² copies of cRNA were tested in duplicate in real-time RT-PCR. A standard curve graph was generated by plotting the C_T values on the y-axis and the log of the input amounts on the x-axis. The slope of this standard curve was −3.43 and the correlation coefficient was 0.997.

Figure 2

The dynamic range of the assay was tested running 10-fold serial dilutions of the authors’ cRNA standard ranging from 10 to 10¹² molecules in the reaction. The amplification plot shows delta Rn on the y-axis (Rn: fluorescence emission intensity of the reporter dye normalized to a passive reference, delta Rn: Rn of an unreacted sample – Rn value of the reaction) against the cycle number displayed on the x-axis. The dynamic range of the assay spans 6 logs ranging from 10² to 10⁸ log molecules of SARS-CoV RNA per reaction, corresponding to C_T values ranging from 37.45 ± 0.04 for 10² copies to 17.40 ± 0.04 for 10⁸ copies. cRNA standards with a copy number above or below the dynamic range are not shown.