Comparison of specific and random priming in the reverse transcriptase polymerase chain reaction for genotyping group A rotaviruses

J Virol Methods. 1999 Mar;78(1-2):93-103. doi: 10.1016/s0166-0934(98)00168-2.

Abstract

This study describes an approach to the molecular typing of rotaviruses which requires only a single RNA extraction and reverse transcription (RT) reaction using random primers. Random-primed RT provides complementary DNA (cDNA) which can be used not only for G- and P-typing polymerase chain reactions (PCR), but also for the detection of other RNA viruses which may act as enteric pathogens. It is a sensitive and specific method that can detect 10 virus particles/ml of 10% faecal suspension provided the cDNA is amplified in a nested typing-PCR. Of 121 specimens positive for rotavirus by EM and analysed using this method, only 8% could not be G- or P-genotyped. The untyped samples were tested again performing the RT reaction with G- and P-specific primers, achieving a 5% increase in sensitivity. Comparing G-genotyping against G-serotyping, 92% were genotyped through random priming RT-PCR whereas only 64% were serotyped using G-serotype specific monoclonal antibodies (MAbs).

Publication types

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

MeSH terms

  • Aged
  • Child
  • DNA Primers
  • DNA, Complementary
  • Diarrhea / virology*
  • Enzyme-Linked Immunosorbent Assay
  • Feces / virology
  • Genotype
  • Humans
  • RNA, Viral / isolation & purification*
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Rotavirus / classification*
  • Rotavirus / genetics*
  • Rotavirus / isolation & purification
  • Rotavirus Infections / virology*
  • Serotyping

Substances

  • DNA Primers
  • DNA, Complementary
  • RNA, Viral