Detection of group B and C rotaviruses by polymerase chain reaction

J Clin Microbiol. 1991 Mar;29(3):519-23. doi: 10.1128/jcm.29.3.519-523.1991.

Abstract

We adapted the polymerase chain reaction (PCR) to detect the noncultivatable group B and C rotaviruses and introduced a simple and convenient technique to purify viral RNA from stool specimens. Double-stranded RNA present in stool extracts was purified by adsorption to hydroxyapatite and was used as the template for reverse transcription and polymerase amplification. Primer pairs specific for group B (gene 8) and group C (gene 6) rotaviruses were selected to amplify group-characteristic sizes of cDNA copies readily identifiable in ethidium bromide-stained agarose gels. These primer pairs were used separately in individual PCR assays or were pooled with a primer pair specific for group A rotavirus (gene 9) in a combined PCR assay for the simultaneous detection of all three rotavirus groups. The method was very sensitive and was used to identify both human and porcine strains of group B and C rotaviruses in stool specimens. A second PCR amplification with internal group-specific primers served to increase further the sensitivity of the test and to confirm the diagnostic results obtained in the first amplification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA, Viral / genetics
  • Genes, Viral
  • Humans
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • RNA, Viral / genetics
  • RNA, Viral / isolation & purification
  • Rotavirus / classification
  • Rotavirus / genetics
  • Rotavirus / isolation & purification*
  • Rotavirus Infections / microbiology
  • Sensitivity and Specificity

Substances

  • DNA, Viral
  • RNA, Viral