Evaluation of advanced reverse transcription-PCR assays and an alternative PCR target region for detection of severe acute respiratory syndrome-associated coronavirus

Abstract

First-generation reverse transcription-PCR (RT-PCR) assays for severe acute respiratory syndrome-associated coronavirus (SARS-CoV) gave false-negative results in a considerable fraction of patients. In the present study, we evaluated two second-generation, replicase (R) gene-based, real-time RT-PCR test kits--the RealArt HPA coronavirus LC kit (Artus, Hamburg, Germany) and the LightCycler SARS-CoV quantification kit (Roche, Penzberg, Germany)--and a real-time RT-PCR assay for the nucleocapsid (N) gene. Detecting the N-gene RNA might be advantageous due to its high abundance in cells. The kits achieved sensitivities of 70.8% (Artus) and 67.1% (Roche) in 66 specimens from patients with confirmed SARS (samples primarily from the upper and lower respiratory tract and stool). The sensitivity of the N-gene assay was 74.2%. The differences in all of the sensitivities were not statistically significant (P = 0.680 [analysis of variance]). Culture cells initially contained five times more N- than R-gene RNA, but the respective levels converged during 4 days of virus replication. In clinical samples the median concentrations of R- and N-gene RNA, respectively, were 1.2 x 10(6) and 2.8 x 10(6) copies/ml (sputum and endotracheal aspirates), 4.3 x 10(4) and 5.5 x 10(4) copies/ml (stool), and 5.5 x 10(2) and 5.2 x 10(2) copies/sample (throat swabs and saliva). Differences between the samples types were significant but not between the types of target RNA. All (n = 12) samples from the lower respiratory tract tested positive in all tests. In conclusion, the novel assays are more sensitive than the first-generation tests, but they still do not allow a comprehensive ruling out of SARS. Methods for the routine sampling of sputum without infection risk are needed to improve SARS RT-PCR.

FIG. 1.

Linear regression of the virus RNA concentrations determined with the N assay (y axis) and each of the two commercial R assays (x axis; suppliers of R assays are identified in the panels) in clinical specimens of SARS patients from Singapore, Hong Kong, and Germany that yielded positive results in both assays (r = correlation coefficient). For swab samples, virus concentrations are expressed in terms of copies per swab; for all other samples, the virus concentration is given as copies per milliliter of sample.

FIG. 2.

Box plot analysis of concentrations of R- and N-gene RNA (y axis) in three different categories of clinical samples (x axis; ETA = endotracheal aspirate). The upper and lower limits of the boxes represent the innermost two quartiles of the ranked datasets, whereas the lines represent the outermost quartiles. Horizontal lines within boxes represent the medians; crosses depict the means of the datasets. The P values associated with each category of samples are derived from Wilcoxon matched-pair signed-rank tests comparing the observed concentrations of the two different RNA species in each category of samples.

FIG. 3.

(A) Absolute concentrations of R- and N-gene RNA in cell culture during the first 4 days after infection (x axis) in cytoplasm (1:100 dilutions of lysates of 10⁶ cells each) and supernatant (copies per 100 μl). (B) Relative amount of N-gene per R-gene RNA in cells and supernatant over time (linear regressions).