Sapovirus detection by quantitative real-time RT-PCR in clinical stool specimens.
Sapovirus (SV) is one of the major causative agents of viral gastroenteritis affecting all age groups worldwide. A new method for the quantitative detection of SV from clinical stool specimens by real-time reverse transcription-polymerase chain reaction (RT-PCR) based on TaqMan MGB technology was described. Primers and probe were designed to target the RNA-dependent RNA polymerase/capsid genes junction. Performance of the newly developed assay was validated against a panel of 244 clinical stool specimens collected for patients with gastroenteritis. SV was detected in eight (3.3%) specimens. Phylogenetic analysis of the positive isolates suggested that the assay could detect at least SV genogroups I, II and IV. In addition, the assay had an increased detection rate compared with a widely used conventional RT-PCR assay. Quantitative analysis showed that the assay could detect as low as 10 copies of viral cDNA per reaction. No cross-reactivity with norovirus and rotavirus was observed. In conclusion, the assay is a sensitive and specific method for the detection of SV from clinical stool specimens.
Chan
Martin C W
MC
Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR.
Sung
Joseph J Y
JJ
Lam
Rebecca K Y
RK
Chan
Paul K S
PK
Lai
Raymond W M
RW
Leung
Wai K
WK
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D017250:Caliciviridae Infections / Q000821:virology*; D002213:Capsid; D002648:Child; D002675:Child, Preschool; D017931:DNA Primers; D005243:Feces / Q000821:virology*; D005260:Female; D005759:Gastroenteritis / Q000821:virology*; D005814:Genes, Viral / Q000235:genetics; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D029341:Sapovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013045:Species Specificity; D010948:Viral Plaque Assay
D017931:DNA Primers; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase