A Real-Time PCR for SARS-coronavirus Incorporating Target Gene Pre-Amplification

Biochem Biophys Res Commun. 2003 Dec 26;312(4):1290-6. doi: 10.1016/j.bbrc.2003.11.064.

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.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Genetic Testing / methods*
  • Genome, Viral
  • Humans
  • Nucleic Acid Amplification Techniques / methods
  • Online Systems
  • Polymerase Chain Reaction / methods*
  • Reproducibility of Results
  • SARS Virus / genetics*
  • SARS Virus / isolation & purification*
  • Sensitivity and Specificity
  • Severe Acute Respiratory Syndrome / diagnosis*
  • Severe Acute Respiratory Syndrome / virology