A real-time PCR for SARS-coronavirus incorporating target gene pre-amplification.
An enhanced polymerase chain reaction (PCR) assay to detect the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) was developed in which a target gene pre-amplification step preceded TaqMan real-time fluorescent PCR. Clinical samples were collected from 120 patients diagnosed as suspected or probable SARS cases and analyzed by conventional PCR followed by agarose gel electrophoresis, conventional TaqMan real-time PCR, and our enhanced TaqMan real-time PCR assays. An amplicon of the size expected from SARS-CoV was obtained from 28/120 samples using the enhanced real-time PCR method. Conventional PCR and real-time PCR alone identified fewer SARS-CoV positive cases. Results were confirmed by viral culture in 3/28 cases. The limit of detection of the enhanced real-time PCR method was 10(2)-fold higher than the standard real-time PCR assay and 10(7)-fold higher than conventional PCR methods. The increased sensitivity of the assay may help control the spread of the disease during future SARS outbreaks.
Lau
Lok Ting
LT
Key Laboratory of Neuroscience, Neuroscience Research Institute, Peking University and Department of Neurobiology, Peking University Health Science Center, 38 Xue Yuan Road, 100083, Beijing, China.
Fung
Yin-Wan Wendy
YW
Wong
Freda Pui-Fan
FP
Lin
Selma Sau-Wah
SS
Wang
Chen Ran
CR
Li
Hui Li
HL
Dillon
Natalie
N
Collins
Richard A
RA
Tam
John Siu-Lun
JS
Chan
Paul K S
PK
Wang
Chen G
CG
Yu
Albert Cheung-Hoi
AC
D005820:Genetic Testing / Q000379:methods*; D016679:Genome, Viral; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009862:Online Systems; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis* / Q000821:virology