• Leo L M Poon, Kwok Hung Chan, On Kei Wong, Wing Cheong Yam, Kwok Yung Yuen, Yi Guan, Y M Dennis Lo, and Joseph S M Peiris.
• Department of Microbiology, Queen Mary Hospital, University of Hong Kong, Pokfulam, Hong Kong. llmpoon@hkucc.hku.hk
• J. Clin. Virol. 2003 Dec 1; 28 (3): 233-8.

BackgroundA 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.ObjectiveTo 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 Design50 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.ResultsOf 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.ConclusionBy 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.

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