title: Development of a novel protocol for RT-multiplex PCR to detect diarrheal viruses among infants and children with acute gastroenteritis in Eastern Russia.
abstract: Viral gastroenteritis is one of the most common illnesses in humans worldwide and it has a great impact on people. Recently, we reported three RT-multiplex PCR assays termed A, B and C that could detect three groups of diarrheal viruses; group A, B and C rotaviruses and adenovirus [Phan et al., J Med Virol 2004; 74:173-9]; norovirus GI and GII, sapovirus and astrovirus [Yan et al., J Virol Meth 2003; 114:37-44]; enteroviruses, hepatitis A and E viruses and influenza A virus [Phan et al., Arch Virol 2005; 1175-85], respectively. In the present study, we developed a novel protocol with a small volume of reaction mixture for RT and PCR products (only 8 microl and 11 microl, respectively) that can amplify genomes of target viruses simultaneously. A total of 100 fecal specimens from infants and children with acute gastroenteritis in Birobiclzhan city, Eastern Russia, were collected during November 2003 and March 2004 and tested for the presence of those viruses by the novel RT-multiplex PCR protocol. Group A rotavirus was the most prevalent (67%), followed by norovirus GII (4%), group C rotavirus (1%), and sapovirus (1%). Interestingly, one fecal specimen turned out to be positive for hepatitis A virus. The sensitivity and specificity of RT-multiplex PCR assays with a novel protocol demonstrated a strong validation against the previously published RT-multiplex PCR. The findings clearly indicated that this novel protocol is simple and cost-effective to investigate the molecular epidemiology of acute gastroenteritis caused by diarrheal viruses. This report is the first, to our knowledge, detecting hepatitis A virus in feces from diarrheal infants and children in Eastern Russia.
authors.affiliation: Department of Developmental Medical Sciences, Institute of International Health, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
authors.forename: Tung Gia
authors.initials: TG
authors.lastname: Phan
authors.forename: Tuan Anh
authors.initials: TA
authors.lastname: Nguyen
authors.forename: Hainian
authors.initials: H
authors.lastname: Yan
authors.forename: Fumihiro
authors.initials: F
authors.lastname: Yagyu
authors.forename: Vladimir
authors.initials: V
authors.lastname: Kozlov
authors.forename: Alexei
authors.initials: A
authors.lastname: Kozlov
authors.forename: Shoko
authors.initials: S
authors.lastname: Okitsu
authors.forename: Werner E G
authors.initials: WE
authors.lastname: Müller
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ushuijma
mesh_terms: D002648:Child; D002675:Child, Preschool; D003967:Diarrhea / Q000821:virology*; D003968:Diarrhea, Infantile / Q000821:virology*; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000821:virology*; D016679:Genome, Viral; D030041:Hepatitis A virus / Q000302:isolation & purification; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D017720:Molecular Epidemiology / Q000379:methods / Q000592:standards; D029322:Norovirus / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000302:isolation & purification; D012426:Russia; D029341:Sapovirus / Q000302:isolation & purification; D012680:Sensitivity and Specificity