title: 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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Microbiology, Queen Mary Hospital, University of Hong Kong, Pokfulam, Hong Kong. llmpoon@hkucc.hku.hk
authors.forename: Leo L M
authors.initials: LL
authors.lastname: Poon
authors.forename: Kwok Hung
authors.initials: KH
authors.lastname: Chan
authors.forename: On Kei
authors.initials: OK
authors.lastname: Wong
authors.forename: Wing Cheong
authors.initials: WC
authors.lastname: Yam
authors.forename: Kwok Yung
authors.initials: KY
authors.lastname: Yuen
authors.forename: Yi
authors.initials: Y
authors.lastname: Guan
authors.forename: Y M Dennis
authors.initials: YM
authors.lastname: Lo
authors.forename: Joseph S M
authors.initials: JS
authors.lastname: Peiris
mesh_terms: D006801:Humans; D009305:Nasopharynx / Q000821:virology; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis* / Q000821:virology; D013997:Time Factors
chemical_list: D012367:RNA, Viral