Detection of norovirus (GI, GII), Sapovirus and astrovirus in fecal samples using reverse transcription single-round multiplex PCR.
A reverse transcription (RT) single-round multiplex polymerase chain reaction (smPCR) assay was developed to detect simultaneously Norovirus genogroup I and II, Sapovirus and astrovirus. A total of 377 diarrhea stool samples (screened for rotavirus- and adenorivus-negative) from four regions in Japan during July 2000 to June 2001 were examined by RT-smPCR. The positive rate was 16.4% (62 out of 377 stool samples). Norovirus, Sapovirus and astrovirus were detected in 42, 16, 4 of 60 positive samples, respectively. Coinfection was not found in these samples. Infections occurred mainly in November, December and January. The key elements of the RT-smPCR are (i) the cDNA synthesis with the Superscript RTII and random primer at 42 degrees C for 1 h, at 99 degrees C for 5 min, and (ii) single-round multiplex PCR by using Taq polymerase mixed together with a mixture of four different primer pairs (G1-SKF/G1-SKR for Norovirus genogroup I, COG2F/G2-SKR for Norovirus genogroup II, SLV5317/SLV5749 for Sapovirus, PreCAP1/82b for astrovirus). All of the four primer pairs amplify the capsid region of target viral genome, produce four size-specific amplicons of 330, 387, 434, 719 bp for Norovirus genogroup I and II, Sapovirus and astrovirus, respectively. This assay provides a more rapid and efficient way to detect these viruses from fecal samples in a single test, and also offers the potential for their molecular detection in food and environmental samples.
Department of Developmental Medical Sciences, Graduate School of Medicine, Institute of International Health, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Hainian
H
Yan
Fumihiro
F
Yagyu
Shoko
S
Okitsu
Osamu
O
Nishio
Hiroshi
H
Ushijima
D000293:Adolescent; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D003967:Diarrhea / Q000821:virology*; D005243:Feces / Q000821:virology*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D001257:Mamastrovirus / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D029322:Norovirus / Q000235:genetics / Q000302:isolation & purification*; D012327:RNA Virus Infections / Q000821:virology; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D029341:Sapovirus / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D012367:RNA, Viral