Increased detection of rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay in stool specimens from children with diarrhea

J Med Virol. 2004 Mar;72(3):496-501. doi: 10.1002/jmv.20009.


Six-hundred and twenty-six stool specimens collected from children with diarrhea over a 12-month period were tested for rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay, a conventional nested PCR assay and by electron microscopy (EM). A fragment of 87 bp in a highly-conserved region of non-structural protein 3 (NSP3) in rotavirus genome was amplified by a single-step RT-PCR protocol in a closed-tube system. Rotavirus was detected in 123 samples (20%) with the real time RT-PCR assay, 113 samples (18%) with the nested-PCR assay, and 79 samples (13%) with EM. Using serial diluted nucleic acid extract, we compared the sensitivity of real time RT-PCR with conventional RT-PCR and conventional nested PCR assays. Real time RT-PCR was two to four logs more sensitive than the conventional assays. The reaction time required for the RT-PCR assay is about half the time required for the conventional nested-PCR. The real time RT-PCR assay is both simple and rapid with advantages including enhanced sensitivity and a lower risk for cross-contamination making it a useful tool for the detection of rotavirus in various situations including sporadic gastroenteritis, outbreaks, and environmental investigations. G(1) was the predominant type (89%), followed by G(2) (10%), and G(4) (1%). No rotavirus of G(3), G(8), and G(9) types were found. The peak season for rotavirus infection was January to May in northern Alberta.

Publication types

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

MeSH terms

  • Canada
  • Child, Preschool
  • Diarrhea / virology*
  • Feces / virology*
  • Genes, Viral / genetics
  • Genotype
  • Humans
  • Microscopy, Electron
  • Molecular Epidemiology
  • RNA, Viral / analysis
  • RNA, Viral / isolation & purification
  • Reverse Transcriptase Polymerase Chain Reaction*
  • Rotavirus / classification
  • Rotavirus / genetics
  • Rotavirus / isolation & purification*
  • Rotavirus Infections / diagnosis*
  • Rotavirus Infections / virology*
  • Sensitivity and Specificity
  • Viral Nonstructural Proteins / genetics


  • NSP3 protein, Rotavirus
  • RNA, Viral
  • Viral Nonstructural Proteins