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 8.4 104 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.

Broadly reactive nested reverse transcription-PCR using an internal RNA standard control for detection of noroviruses in stool samples / Medici, Maria Cristina; Martinelli, M.; Ruggeri, F. M.; Abelli, L. A.; Bosco, S.; Arcangeletti, Maria Cristina; Pinardi, F.; DE CONTO, Flora; Calderaro, Adriana; Chezzi, Carlo; Dettori, Giuseppe. - In: JOURNAL OF CLINICAL MICROBIOLOGY. - ISSN 0095-1137. - 43(8):(2005), pp. 3772-3778. [10.1128/JCM.43.8.3772-3778.2005]

Broadly reactive nested reverse transcription-PCR using an internal RNA standard control for detection of noroviruses in stool samples

MEDICI, Maria Cristina;ARCANGELETTI, Maria Cristina;DE CONTO, Flora;CALDERARO, Adriana;CHEZZI, Carlo;DETTORI, Giuseppe
2005-01-01

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 8.4 104 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.
2005
Broadly reactive nested reverse transcription-PCR using an internal RNA standard control for detection of noroviruses in stool samples / Medici, Maria Cristina; Martinelli, M.; Ruggeri, F. M.; Abelli, L. A.; Bosco, S.; Arcangeletti, Maria Cristina; Pinardi, F.; DE CONTO, Flora; Calderaro, Adriana; Chezzi, Carlo; Dettori, Giuseppe. - In: JOURNAL OF CLINICAL MICROBIOLOGY. - ISSN 0095-1137. - 43(8):(2005), pp. 3772-3778. [10.1128/JCM.43.8.3772-3778.2005]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11381/1444276
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