A real-time PCR for SARS-coronavirus incorporating target gene pre-amplification

• Published in: Biochemical and biophysical research communications Vol. 312; no. 4; pp. 1290 - 1296
• Main Authors: Lau, Lok Ting, Fung, Yin-Wan Wendy, Wong, Freda Pui-Fan, Lin, Selma Sau-Wah, Wang, Chen Ran, Li, Hui Li, Dillon, Natalie, Collins, Richard A, Tam, John Siu-Lun, Chan, Paul K.S, Wang, Chen G, Yu, Albert Cheung-Hoi
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 26.12.2003
• Subjects: Coronavirus; Genetic Testing - methods; Genome, Viral; Humans; Nucleic Acid Amplification Techniques - methods; Online Systems; PCR; Polymerase Chain Reaction - methods; Reproducibility of Results; SARS; SARS coronavirus; SARS Virus - genetics; SARS Virus - isolation & purification; SARS-CoV; Sensitivity and Specificity; Severe acute respiratory syndrome; Severe Acute Respiratory Syndrome - diagnosis; Severe Acute Respiratory Syndrome - virology; TaqMan real-time fluorescent polymerase chain reaction; SARS; Coronavirus; TaqMan real-time fluorescent polymerase chain reaction; Severe acute respiratory syndrome; PCR; SARS-CoV
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2003.11.064

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.