Development of a molecular-beacon-based multi-allelic real-time RT-PCR assay for the detection of human coronavirus causing severe acute respiratory syndrome (SARS-CoV): a general methodology for detecting rapidly mutating viruses

Arch Virol. 2011 Apr;156(4):671-80. doi: 10.1007/s00705-010-0906-7. Epub 2011 Jan 11.

Abstract

Emerging infectious diseases have caused a global effort for development of fast and accurate detection techniques. The rapidly mutating nature of viruses presents a major difficulty, highlighting the need for specific detection of genetically diverse strains. One such infectious agent is SARS-associated coronavirus (SARS-CoV), which emerged in 2003. This study aimed to develop a real-time RT-PCR detection assay specific for SARS-CoV, taking into account its intrinsic polymorphic nature due to genetic drift and recombination and the possibility of continuous and multiple introductions of genetically non-identical strains into the human population, by using mismatch-tolerant molecular beacons designed to specifically detect the SARS-CoV S, E, M and N genes. These were applied in simple, reproducible duplex and multiplex real-time PCR assays on 25 post-mortem samples and constructed RNA controls, and they demonstrated high target detection ability and specificity. This assay can readily be adapted for detection of other emerging and rapidly mutating pathogens.

Publication types

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

MeSH terms

  • Humans
  • Molecular Diagnostic Techniques / methods*
  • Mutation
  • Oligonucleotide Probes / chemistry
  • Oligonucleotide Probes / genetics
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • SARS Virus / genetics
  • SARS Virus / isolation & purification*
  • Sensitivity and Specificity
  • Severe Acute Respiratory Syndrome / diagnosis*
  • Virology / methods*

Substances

  • Oligonucleotide Probes