title: Broadly reactive nested reverse transcription-PCR using an internal RNA standard control for detection of noroviruses in stool samples.
abstract: We developed a nested reverse transcription-PCR (nRT-PCR) for the detection of noroviruses in stools, using random primers for RT, the JV12/JV13 primer pair in the first round of nPCR, and a set of nine inner primers for the second, comprising the reverse sequences of primers SR46, SR48, SR50, and SR52, and five novel oligonucleotide sequences (113-1, 113-2, 115-1, 115-2, and 115-3). The specificity of the nRT-PCR was confirmed by testing 61 stools containing enteric viruses other than noroviruses. In comparative assays on either stools or RNA dilutions from two genogroup I and three genogroup II (GII) norovirus-positive samples, nRT-PCR was always at least as sensitive as RT-PCR and Southern hybridization. With some of the samples tested, the increase in sensitivity was 10-fold or higher. For GII viruses, the detectable range of nRT-PCR was estimated to be 8.4 x 10(4) to 2 RNA viral particles. When used on 85 stools from pediatric patients with acute gastroenteritis negative for viruses by electron microscopy and cell culture, the nRT-PCR detected norovirus in 19 samples (22.3%), while it failed to detect one reference RT-PCR-positive sample containing a Desert Shield strain. Sixteen of the 19 nRT-PCR-positive samples gave concordant results with reference RT-PCR and Southern hybridization, and all with sequence analysis. Partial sequencing of the polymerase region revealed that from January to April 2000 all GII strains except two (Rotterdam- and Leeds-like viruses) formed a tight cluster related to Hawaii virus. The nRT-PCR described could prove suitable for large epidemiological studies and for specialized clinical laboratories performing routine molecular testing.
authors.affiliation: Section of Microbiology, Department of Pathology and Laboratory Medicine, University of Parma, Viale Antonio Gramsci, 14, 43100 Parma, Italy. mariacristina.medici@unipr.it
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Medici
authors.forename: Monica
authors.initials: M
authors.lastname: Martinelli
authors.forename: Franco Maria
authors.initials: FM
authors.lastname: Ruggeri
authors.forename: Laura Anna
authors.initials: LA
authors.lastname: Abelli
authors.forename: Simona
authors.initials: S
authors.lastname: Bosco
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Arcangeletti
authors.forename: Federica
authors.initials: F
authors.lastname: Pinardi
authors.forename: Flora
authors.initials: F
authors.lastname: De Conto
authors.forename: Adriana
authors.initials: A
authors.lastname: Calderaro
authors.forename: Carlo
authors.initials: C
authors.lastname: Chezzi
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Dettori
mesh_terms: D002648:Child; D002675:Child, Preschool; D005243:Feces / Q000821:virology*; D006801:Humans; D007223:Infant; D008854:Microscopy, Electron; D029322:Norovirus / Q000235:genetics / Q000302:isolation & purification*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA