Early diagnosis of SARS coronavirus infection by real time RT-PCR

Abstract

Background: A novel coronavirus was recently identified as the aetiological agent of Severe Acute Respiratory Syndrome (SARS). Molecular assays currently available for detection of SARS-coronavirus (SARS-CoV) have low sensitivity during the early stage of the illness.

Objective: To develop and evaluate a sensitive diagnostic test for SARS by optimizing the viral RNA extraction methods and by applying real-time quantitative RT-PCR technology.

Study design: 50 nasopharyngeal aspirate (NPA) samples collected from days 1-3 of disease onset from SARS patients in whom SARS CoV infections was subsequently serologically confirmed and 30 negative control samples were studied. Samples were tested by: (1) our first generation conventional RT-PCR assay with a routine RNA extraction method (Lancet 361 (2003) 1319), (2) our first generation conventional RT-PCR assay with a modified RNA extraction method, (3) a real-time quantitative RT-PCR assay with a modified RNA extraction method.

Results: Of 50 NPA specimens collected during the first 3 days of illness, 11 (22%) were positive in our first generation RT-PCR assay. With a modification in the RNA extraction protocol, 22 (44%) samples were positive in the conventional RT-PCR assay. By combining the modified RNA extraction method and real-time quantitative PCR technology, 40 (80%) of these samples were positive in the real-time RT-PCR assay. No positive signal was observed in the negative controls.

Conclusion: By optimizing RNA extraction methods and applying quantitative real time RT-PCR technologies, the sensitivity of tests for early diagnosis of SARS can be greatly enhanced.

Fig. 1

Real-time quantitative RT-PCR assays for SARS-CoV. (A) Standard curve for quantitative analysis of ORF 1b of SARS-CoV. The threshold cycle (Ct) is the number of PCR cycles required for the fluorescent intensity of the reaction to reach a pre-defined threshold. The Ct is inversely proportional to the logarithm of the starting concentration of the input DNA. (B) An amplification plot of fluorescence intensity against the PCR cycle. The amplification curves of positive clinical samples, negative clinical samples and water controls are indicated. The X axis denotes the cycle number of a quantitative PCR assay. The Y axis denotes the fluorescence intensity over the background.

Fig. 2

Changes of viral loads of SARS-CoV in NPA samples collected at day 1-3 of disease onset. Open bar: viral loads of SARS-CoV from samples that were positive in the real-time quantitative PCR assay. Grey bar: Viral loads of SARS-Cov from samples that were positive in the real-time assay, but negative in the conventional RT-PCR assay. The upper and lower limits of the boxes and the lines across the boxes indicate the 75th and 25th percentiles and the median, respectively. The upper and lower horizontal bars indicate the 90th and 10th percentiles, respectively.