Development of a quantitative real-time PCR assay for sapovirus in children under 5-years-old in Regina Margherita Hospital of Turin, Italy.
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.
a Department of Public Health and Pediatric Sciences, University of Turin, Medical School, 10136 Turin, Italy.
Massimiliano
M
Bergallo
a Department of Public Health and Pediatric Sciences, University of Turin, Medical School, 10136 Turin, Italy.
Ilaria
I
Galliano
a Department of Public Health and Pediatric Sciences, University of Turin, Medical School, 10136 Turin, Italy.
Paola
P
Montanari
b Struttura Complessa, Pediatric University Regina Margherita Hospital, Piazza Polonia 94, 1016 Turin, Italy.
Martina Rosa
MR
Brusin
a Department of Public Health and Pediatric Sciences, University of Turin, Medical School, 10136 Turin, Italy.
Serena
S
Finotti
a Department of Public Health and Pediatric Sciences, University of Turin, Medical School, 10136 Turin, Italy.
Giulia
G
Paderi
b Struttura Complessa, Pediatric University Regina Margherita Hospital, Piazza Polonia 94, 1016 Turin, Italy.
Clara
C
Gabiano
D002675:Child, Preschool; D005243:Feces / Q000821:virology; D005260:Female; D005759:Gastroenteritis / Q000821:virology*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D029341:Sapovirus / Q000302:isolation & purification*; D019562:Viral Load
D012367:RNA, Viral
PCR en temps réel; RNA extraction; Sapovirus; co-infection; coïnfection; extraction d’ARN; gastroenteritis; gastroentérite; real-time PCR