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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.

SUBMITTER: Lau LT 

PROVIDER: S-EPMC7111096 | biostudies-literature | 2003 Dec

REPOSITORIES: biostudies-literature

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

Lau Lok Ting LT   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  

Biochemical and biophysical research communications 20031201 4


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 amplic  ...[more]

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