Comparison of two real-time quantitative assays for detection of severe acute respiratory syndrome coronavirus

Abstract

The new severe acute respiratory syndrome (SARS) coronavirus (CoV), described in February 2003, infected a total of 8,439 people. A total of 812 people died due to respiratory insufficiency. Close contact with symptomatic patients appeared to be the main route of transmission. However, potential transmission by blood transfusion could not be definitely excluded. Two real-time SARS-specific PCR assays were assessed for their sensitivities, agreement of test results, and intra-assay variabilities. Both assays rely on reverse transcription and amplification of extracted RNA. Dilutions of gamma-irradiated cell culture supernatants of SARS CoV-infected Vero E6 cells were prepared to determine the precisions, linear ranges, and accuracies of the assays. The linear range for the Artus RealArt HPA-Coronavirus assay (Artus assay) was 1 x 10(2) to 1 x 10(7) copies/ml, and that for the Roche LightCycler SARS CoV Quantification kit (Roche assay) was 1 x 10(4) to 2 x 10(8) copies/ml. The detection limit of the Roche assay was 3,982.1 copies/ml, whereas that of the Artus assay was 37.8 copies/ml. Detection limits were calculated with a standard preparation that was recommended for use by the World Health Organization. However, quantification of CoV in this preparation may be imprecise. In summary, both assays are suitable for quantitative measurement of SARS CoV at the high concentrations expected in sputum samples. The Artus assay is also suitable for detection of SARS CoV at the low concentrations found in serum samples.

FIG. 1.

Linear ranges of SARS CoV assays. (A) Correlation between nominal SARS CoV RNA concentrations and measured SARS RNA concentrations analyzed with the external standard RNA. ▴, Roche assay; ▪, Artus assay; •, line of equality. (B) Correlation between nominal and measured SARS CoV RNA concentrations analyzed with kit-specific internal standards. The correlation factors (R² values) showed no significant differences.

FIG. 1.

Linear ranges of SARS CoV assays. (A) Correlation between nominal SARS CoV RNA concentrations and measured SARS RNA concentrations analyzed with the external standard RNA. ▴, Roche assay; ▪, Artus assay; •, line of equality. (B) Correlation between nominal and measured SARS CoV RNA concentrations analyzed with kit-specific internal standards. The correlation factors (R² values) showed no significant differences.

FIG. 2.

Real-time PCR of SARS CoV by two real-time PCRs: the Roche assay (I) and the Artus assay (II). Real-time PCR runs for SARS CoV with six external standard RNA concentrations (A, 10⁶ copies/ml; B, 10⁵ copies/ml; C, 10⁴ copies/ml; D, 10³ copies/ml; E, 10² copies/ml; F, 10¹ copies/ml; NTC, no-template control) are shown. The Roche assay demonstrates positive results only for the first five concentrations (A to E), whereas the Artus assay shows positive results for all six concentrations (A to F).

FIG. 3.

Agreement between Roche assay and Artus assay. The results for external standard RNAs (10⁶ to 10¹ copies/ml) were plotted against each other. The line of equality is represented by a dotted line. ▪, measured values.

FIG. 4.

Limits of agreement according to Bland and Altman (4). The means of both methods (x axis) were plotted against the differences between the means of the two methods (y axis). (A) External standard RNA concentration of 10⁶ copies/ml; (B) external standard RNA concentration of 10⁴ copies/ml; (C) external standard RNA concentration of 10² copies/ml. The mean is indicated by a solid line; the means ± two times the SD (2SD) are indicated by dotted lines.