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

J Clin Virol. 2003 Dec;28(3):233-8. doi: 10.1016/j.jcv.2003.08.004.

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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Humans
  • Nasopharynx / virology
  • RNA, Viral / analysis
  • RNA, Viral / isolation & purification
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • SARS Virus / isolation & purification*
  • Sensitivity and Specificity
  • Severe Acute Respiratory Syndrome / diagnosis*
  • Severe Acute Respiratory Syndrome / virology
  • Time Factors

Substances

  • RNA, Viral