Real-time reverse transcription PCR detection of norovirus, sapovirus and astrovirus as causative agents of acute viral gastroenteritis

Abstract

The design and development of highly sensitive real-time reverse transcription PCR assays for the detection of norovirus genogroups I, II and IV, sapovirus genogroups I, II and IV, and human astrovirus from stool samples is described. Examination of 140 stool samples from paediatric patients exhibiting symptoms of diarrhoea and/or vomiting resulted in increased detection levels as compared to examination by electron microscopy. Real-time PCR resulted in a 200% increase in the rate of detection of norovirus as compared to electron microscopy. Only genogroup II noroviruses were detected in the stool specimens and when examined using partial-genotyping primers all were identified as clustering with the genogroup II/4 (Bristol/Lordsdale) cluster. Sapovirus was not detected in any of the stool specimens by electron microscopy while 11% (15/140) of specimens were sapovirus positive by real-time RT-PCR, accounting for 36% of calicivirus diarrhoea. Real-time RT-PCR resulted in a tenfold increase in the rate of detection of astrovirus when compared to detection by electron microscopy with both type 1 and type 4 human astroviruses being detected in circulation. The results highlight the importance of the introduction of molecular methods for the routine screening of stool samples for causative agents of viral gastroenteritis.