Increased detection of rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay in stool specimens from children with diarrhea.
Six-hundred and twenty-six stool specimens collected from children with diarrhea over a 12-month period were tested for rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay, a conventional nested PCR assay and by electron microscopy (EM). A fragment of 87 bp in a highly-conserved region of non-structural protein 3 (NSP3) in rotavirus genome was amplified by a single-step RT-PCR protocol in a closed-tube system. Rotavirus was detected in 123 samples (20%) with the real time RT-PCR assay, 113 samples (18%) with the nested-PCR assay, and 79 samples (13%) with EM. Using serial diluted nucleic acid extract, we compared the sensitivity of real time RT-PCR with conventional RT-PCR and conventional nested PCR assays. Real time RT-PCR was two to four logs more sensitive than the conventional assays. The reaction time required for the RT-PCR assay is about half the time required for the conventional nested-PCR. The real time RT-PCR assay is both simple and rapid with advantages including enhanced sensitivity and a lower risk for cross-contamination making it a useful tool for the detection of rotavirus in various situations including sporadic gastroenteritis, outbreaks, and environmental investigations. G(1) was the predominant type (89%), followed by G(2) (10%), and G(4) (1%). No rotavirus of G(3), G(8), and G(9) types were found. The peak season for rotavirus infection was January to May in northern Alberta.
Pang
Xiaoli L
XL
Provincial Laboratory for Public Health (Microbiology), University Alberta Hospital, Edmonton, Alberta, Canada. x.pang@provlab.ab.ca
Lee
Bonita
B
Boroumand
Nasim
N
Leblanc
Barbara
B
Preiksaitis
Jutta K
JK
Yu Ip
Charlotte C
CC
D002170:Canada; D002675:Child, Preschool; D003967:Diarrhea / Q000821:virology*; D005243:Feces / Q000821:virology*; D005814:Genes, Viral / Q000235:genetics; D005838:Genotype; D006801:Humans; D008854:Microscopy, Electron; D017720:Molecular Epidemiology; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012401:Rotavirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000175:diagnosis* / Q000821:virology*; D012680:Sensitivity and Specificity; D017361:Viral Nonstructural Proteins / Q000235:genetics
C082740:NSP3 protein, Rotavirus; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins