Development and application of a capsid VP1 (region D) based reverse transcription PCR assay for genotyping of genogroup I and II noroviruses

Noroviruses (NoV), previously called “Norwalk-like viruses”, have emerged as the single most important cause of acute gastroenteritis worldwide. Most diagnostic reverse transcription-polymerase chain reaction (RT-PCR) assays target the viral RNA-dependent RNA polymerase; however, the major capsid pr...

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Bibliographic Details
Published in:Journal of virological methods 2004-03, Vol.116 (2), p.109-117
Main Authors: Vinjé, Jan, Hamidjaja, Raditijo A., Sobsey, Mark D.
Format: Article
Language:English
Subjects:
Acute gastroenteritis
Base Sequence
Biological and medical sciences
Capsid
Capsid Proteins - genetics
DNA Primers
Fundamental and applied biological sciences. Psychology
Genotype
Genotyping
Microbiology
norovirus
Norovirus - classification
Norovirus - genetics
Noroviruses
Phylogeny
Reverse Transcriptase Polymerase Chain Reaction - methods
RNA, Viral - genetics
RNA, Viral - isolation & purification
Techniques used in virology
Virology
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Summary:Noroviruses (NoV), previously called “Norwalk-like viruses”, have emerged as the single most important cause of acute gastroenteritis worldwide. Most diagnostic reverse transcription-polymerase chain reaction (RT-PCR) assays target the viral RNA-dependent RNA polymerase; however, the major capsid protein (VP1) is the reference genomic region for establishing genotypes. In this study, we analyzed complete NoV VP1 sequences ( n=100) and determined a region (region D) that was most suitable to differentiate between genotypes. Within region D, we designed two genogroup specific, broadly reactive, degenerate primer sets (GI and GII). The region D primers were evaluated in a single-tube one-step RT-PCR assay using a panel of 81 (31 GI, 50 GII) NoV strains from both outbreaks and sporadic cases. In total, 95% of the samples tested positive using the new region D primer sets. Phylogenetic analysis of region D sequences (36 deduced amino acids for GI, 56 deduced amino acids for GII), revealed 19 clusters (7 within GI and 12 within GII) including three new genetically distinct clusters, two of which were unresolved using region A sequences. Phylogenetic analysis of the complete VP1 sequences revealed identical grouping of strains and confirmed the newly identified clusters using region D. In summary, we successfully developed and evaluated a broadly reactive RT-PCR assay for reliable genotyping of GI and GII noroviruses.
ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2003.11.001