Broadly reactive nested reverse transcription-PCR using an internal RNA standard control for detection of noroviruses in stool samples.
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.
Medici
Maria Cristina
MC
Section of Microbiology, Department of Pathology and Laboratory Medicine, University of Parma, Viale Antonio Gramsci, 14, 43100 Parma, Italy. mariacristina.medici@unipr.it
Martinelli
Monica
M
Ruggeri
Franco Maria
FM
Abelli
Laura Anna
LA
Bosco
Simona
S
Arcangeletti
Maria Cristina
MC
Pinardi
Federica
F
De Conto
Flora
F
Calderaro
Adriana
A
Chezzi
Carlo
C
Dettori
Giuseppe
G
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