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.
Yan
Hainian
H
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.
Yagyu
Fumihiro
F
Okitsu
Shoko
S
Nishio
Osamu
O
Ushijima
Hiroshi
H
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