Gastroenteritis is a common disease in children, characterized by diarrhea, vomiting, abdominal pain, and fever. Sapovirus (SaV) is a causative agent of acute gastroenteritis although it causes milder illness than rotavirus and norovirus. There is high variability in analytical performance of quantitative PCR-based assays amongst clinical laboratories. This study develops a RT real-time PCR method to detect SaV in fecal specimens collected from under-five-year-old children with acute gastroenteritis. 15 out of 137 (10.9%) episodes of acute gastroenteritis were associated with SaV genomic detection with median viral load 6.6(log10) ± 7.1(log10) genomes/mg fecal specimens. There was significant difference in detection rate between male and female (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 manual RNAzol protocol and 31 to 90% in automated Maxwell. We also studied whether human genomic DNA influence 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 stool of children with acute gastroenteritis, are described.
Development of a quantitative real-time PCR assay for sapovirus in children under 5-years-old in Regina Margherita Hospital of Turin, Italy
BERGALLO, Massimiliano
First
;GALLIANO, Ilaria;MONTANARI, Paola;Rosa-Brusin, Martina;GABIANO, CLARA
2017-01-01
Abstract
Gastroenteritis is a common disease in children, characterized by diarrhea, vomiting, abdominal pain, and fever. Sapovirus (SaV) is a causative agent of acute gastroenteritis although it causes milder illness than rotavirus and norovirus. There is high variability in analytical performance of quantitative PCR-based assays amongst clinical laboratories. This study develops a RT real-time PCR method to detect SaV in fecal specimens collected from under-five-year-old children with acute gastroenteritis. 15 out of 137 (10.9%) episodes of acute gastroenteritis were associated with SaV genomic detection with median viral load 6.6(log10) ± 7.1(log10) genomes/mg fecal specimens. There was significant difference in detection rate between male and female (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 manual RNAzol protocol and 31 to 90% in automated Maxwell. We also studied whether human genomic DNA influence 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 stool of children with acute gastroenteritis, are described.
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