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

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.

Detection of SARS coronavirus in patients with severe acute respiratory syndrome by conventional and real-time quantitative reverse transcription-PCR assays.

BACKGROUND: A novel coronavirus (CoV) was recently identified as the agent for severe acute respiratory syndrome (SARS). We compared the abilities of conventional and real-time reverse transcription-PCR (RT-PCR) assays to detect SARS CoV in clinical specimens. METHODS: RNA samples isolated from nasopharyngeal aspirate (NPA; n = 170) and stool (n = 44) were reverse-transcribed and tested by our in-house conventional RT-PCR assay. We selected 98 NPA and 37 stool samples collected at different times after the onset of disease and tested them in a real-time quantitative RT-PCR specific for the open reading frame (ORF) 1b region of SARS CoV. Detection rates for the conventional and real-time quantitative RT-PCR assays were compared. To investigate the nature of viral RNA molecules in these clinical samples, we determined copy numbers of ORF 1b and nucleocapsid (N) gene sequences of SARS CoV. RESULTS: The quantitative real-time RT-PCR assay was more sensitive than the conventional RT-PCR assay for detecting SARS CoV in samples collected early in the course of the disease. Real-time assays targeted at the ORF 1b region and the N gene revealed that copy numbers of ORF 1b and N gene sequences in clinical samples were similar. CONCLUSIONS: NPA and stool samples can be used for early diagnosis of SARS. The real-time quantitative RT-PCR assay for SARS CoV is potentially useful for early detection of SARS CoV. Our results suggest that genomic RNA is the predominant viral RNA species in clinical samples.