title: A real-time PCR for SARS-coronavirus incorporating target gene pre-amplification.
abstract: 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.
authors.affiliation: 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.
authors.forename: Lok Ting
authors.initials: LT
authors.lastname: Lau
authors.forename: Yin-Wan Wendy
authors.initials: YW
authors.lastname: Fung
authors.forename: Freda Pui-Fan
authors.initials: FP
authors.lastname: Wong
authors.forename: Selma Sau-Wah
authors.initials: SS
authors.lastname: Lin
authors.forename: Chen Ran
authors.initials: CR
authors.lastname: Wang
authors.forename: Hui Li
authors.initials: HL
authors.lastname: Li
authors.forename: Natalie
authors.initials: N
authors.lastname: Dillon
authors.forename: Richard A
authors.initials: RA
authors.lastname: Collins
authors.forename: John Siu-Lun
authors.initials: JS
authors.lastname: Tam
authors.forename: Paul K S
authors.initials: PK
authors.lastname: Chan
authors.forename: Chen G
authors.initials: CG
authors.lastname: Wang
authors.forename: Albert Cheung-Hoi
authors.initials: AC
authors.lastname: Yu
mesh_terms: 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