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

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

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.