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.


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.
Poon
Leo L M
LL
Department of Microbiology, Queen Mary Hospital, University of Hong Kong, Pokfulam, Hong Kong. llmpoon@hkucc.hku.hk
Chan
Kwok Hung
KH
Wong
On Kei
OK
Yam
Wing Cheong
WC
Yuen
Kwok Yung
KY
Guan
Yi
Y
Lo
Y M Dennis
YM
Peiris
Joseph S M
JS
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
D012367:RNA, Viral