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. 2017 Apr;63(4):296-302.
doi: 10.1139/cjm-2016-0482. Epub 2016 Dec 2.

Development of a quantitative real-time PCR assay for sapovirus in children under 5-years-old in Regina Margherita Hospital of Turin, Italy

Massimiliano Bergallo  1 , Ilaria Galliano  1 , Paola Montanari  1 , Martina Rosa Brusin  2 , Serena Finotti  1 , Giulia Paderi  1 , Clara Gabiano  2
Affiliations

Development of a quantitative real-time PCR assay for sapovirus in children under 5-years-old in Regina Margherita Hospital of Turin, Italy

Massimiliano Bergallo et al. Can J Microbiol. 2017 Apr.

Abstract

Gastroenteritis is a common disease in children. It is characterized by diarrhea, vomiting, abdominal pain, and fever. Sapovirus (SaV) is a causative agent of acute gastroenteritis, but it causes milder illness than do rotavirus and norovirus. There is high variability in the analytical performance of quantitative PCR-based assays among clinical laboratories. This study developed a reverse transcription real-time PCR method to detect SaV in fecal specimens collected from children under 5-years-old with acute gastroenteritis. Of 137 episodes of acute gastroenteritis, 15 (10.9%) were associated with SaV genomic detection, with a median viral load of 6.6(log10) ± 7.1(log10) genomes/mg fecal specimens. There was a significant difference in detection rate between males and females (9.48% (13/15) vs. 1.46% (2/15), p = 0.0232). Among the 15 SaV-positive cases, 6 were also positive for rotavirus. Viral RNA recovery rate ranged from 46% to 77% in the manual RNAzol protocol and from 31% to 90% in the automated Maxwell protocol. We also studied whether human genomic DNA influences the sensitivity of the assay: its presence caused a decrease in PCR sensitivity. The development of a laboratory-designed real-time PCR TaqMan assay for quantitative detection of SaV and the optimization and standardization of this assay, using stools of children with acute gastroenteritis, are described.

Keywords: PCR en temps réel; RNA extraction; Sapovirus; co-infection; coïnfection; extraction d’ARN; gastroenteritis; gastroentérite; real-time PCR.

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