Detection of norovirus (GI, GII), Sapovirus and astrovirus in fecal samples using reverse transcription single-round multiplex PCR

J Virol Methods. 2003 Dec;114(1):37-44. doi: 10.1016/j.jviromet.2003.08.009.


A reverse transcription (RT) single-round multiplex polymerase chain reaction (smPCR) assay was developed to detect simultaneously Norovirus genogroup I and II, Sapovirus and astrovirus. A total of 377 diarrhea stool samples (screened for rotavirus- and adenorivus-negative) from four regions in Japan during July 2000 to June 2001 were examined by RT-smPCR. The positive rate was 16.4% (62 out of 377 stool samples). Norovirus, Sapovirus and astrovirus were detected in 42, 16, 4 of 60 positive samples, respectively. Coinfection was not found in these samples. Infections occurred mainly in November, December and January. The key elements of the RT-smPCR are (i) the cDNA synthesis with the Superscript RTII and random primer at 42 degrees C for 1 h, at 99 degrees C for 5 min, and (ii) single-round multiplex PCR by using Taq polymerase mixed together with a mixture of four different primer pairs (G1-SKF/G1-SKR for Norovirus genogroup I, COG2F/G2-SKR for Norovirus genogroup II, SLV5317/SLV5749 for Sapovirus, PreCAP1/82b for astrovirus). All of the four primer pairs amplify the capsid region of target viral genome, produce four size-specific amplicons of 330, 387, 434, 719 bp for Norovirus genogroup I and II, Sapovirus and astrovirus, respectively. This assay provides a more rapid and efficient way to detect these viruses from fecal samples in a single test, and also offers the potential for their molecular detection in food and environmental samples.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Base Sequence
  • Child
  • Child, Preschool
  • Diarrhea / virology*
  • Feces / virology*
  • Humans
  • Infant
  • Infant, Newborn
  • Mamastrovirus / genetics
  • Mamastrovirus / isolation & purification*
  • Molecular Sequence Data
  • Norovirus / genetics
  • Norovirus / isolation & purification*
  • RNA Virus Infections / virology
  • RNA, Viral / analysis
  • RNA, Viral / isolation & purification
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Sapovirus / genetics
  • Sapovirus / isolation & purification*
  • Sensitivity and Specificity


  • RNA, Viral