Sensitive and rapid detection of norovirus using duplex TaqMan reverse transcription-polymerase chain reaction.
Conventional reverse transcription-polymerase chain reaction (RT-PCR) to detect norovirus (NV) is a complex of multi-step procedure that requires gel electrophoresis as well as hybridization or sequencing to confirm the final diagnosis. A duplex TaqMan RT-PCR was developed to detect and classify genogroup (G) I and GII of NV. The primers and TaqMan probes for this assay were selected from the region of open reading frame (ORF) 1-ORF2 junction. A total of 796 stool specimens from 103 outbreaks of gastroenteritis, and a series of 46 stool specimens containing most NV genotypes was used for this study. For these specimens from 103 outbreaks, NV was detected and classified by the duplex TaqMan RT-PCR in 536 of the 541 specimens tested previously to be positive using the conventional RT-PCR. Two hundred fifty-one of the 255 specimens that were negative by the conventional RT-PCR were also negative by the TaqMan RT-PCR. No false positive result was observed for other enteric RNA viruses such as rotavirus and sapovirus. This is the first report on the development of a duplex TaqMan RT-PCR end-point assay for detection and differentiation of GI and GII of NV strains simultaneously followed by genotyping. These results suggest the practical application of this duplex TaqMan RT-PCR is useful for the detection of NV in clinical specimens.
Ishida
Setsuko
S
Division of Enteric Virology, Department of Microbiology, Hokkaido Institute of Public Health, Sapporo, Japan. ishidas@iph.pref.hokkaido.jp
Yoshizumi
Shima
S
Ikeda
Tetsuya
T
Miyoshi
Masahiro
M
Okano
Motohiko
M
Okui
Toyo
T
D001483:Base Sequence; D017250:Caliciviridae Infections / Q000175:diagnosis* / Q000453:epidemiology; D017931:DNA Primers / Q000235:genetics; D004196:Disease Outbreaks; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000453:epidemiology / Q000821:virology*; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D029322:Norovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
D017931:DNA Primers; D012367:RNA, Viral