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Single-step RT-PCR assay for dual genotyping of GI and GII norovirus strains
BACKGROUND: Noroviruses are the major cause of acute gastroenteritis (AGE) in people of all ages globally. Standardized genotyping is key for outbreak investigations and surveillance networks. OBJECTIVE: Here we describe the validation of a one-step conventional RT-PCR assay for sequence-based dual...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier Science
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816162/ https://www.ncbi.nlm.nih.gov/pubmed/33260046 http://dx.doi.org/10.1016/j.jcv.2020.104689 |
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author | Chhabra, Preeti Browne, Hannah Huynh, Thalia Diez-Valcarce, Marta Barclay, Leslie Kosek, Margaret N. Ahmed, Tahmeed Lopez, Maria Renee Pan, Chao-Yang Vinjé, Jan |
author_facet | Chhabra, Preeti Browne, Hannah Huynh, Thalia Diez-Valcarce, Marta Barclay, Leslie Kosek, Margaret N. Ahmed, Tahmeed Lopez, Maria Renee Pan, Chao-Yang Vinjé, Jan |
author_sort | Chhabra, Preeti |
collection | PubMed |
description | BACKGROUND: Noroviruses are the major cause of acute gastroenteritis (AGE) in people of all ages globally. Standardized genotyping is key for outbreak investigations and surveillance networks. OBJECTIVE: Here we describe the validation of a one-step conventional RT-PCR assay for sequence-based dual typing of GI and GII noroviruses. This polymerase (P) and capsid (C) dual typing assay uses a combination of previously published oligonucleotide primers amplifying a genomic region spanning the 3'-end of ORF1 and 5'end of ORF2 resulting in a 579 bp product for GI and 570 bp product for GII viruses. RESULTS: The limit of detection of the assay ranged from 5 to 50 copies of viral RNA per reaction for GI and GII. To validate the assay, we tested 2,663 noroviruspositive stool samples from outbreaks and sporadic cases of AGE in Bangladesh, Guatemala, Peru, and USA collected between 2010–2019, of which 2,392 (90 %) were genotyped successfully. Most of the known genotypes infecting humans (GI (n = 9) and GII (n = 23)) and P types (GI (n = 15), GII, (n = 20)) could be detected. The remaining 270 samples had low viral load (Ct > 30) by real-time RT-PCR. A panel of 166 samples positive for other enteric viruses (rotavirus, astrovirus, sapovirus, adenovirus type 40/41) tested negative. CONCLUSION: The use of broadly reactive genotyping assays greatly strengthens exchange of standardized genotype data globally to monitor trends in genotype diversity which is important for both the development of vaccines and to measure their impact. |
format | Online Article Text |
id | pubmed-7816162 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Elsevier Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-78161622021-01-29 Single-step RT-PCR assay for dual genotyping of GI and GII norovirus strains Chhabra, Preeti Browne, Hannah Huynh, Thalia Diez-Valcarce, Marta Barclay, Leslie Kosek, Margaret N. Ahmed, Tahmeed Lopez, Maria Renee Pan, Chao-Yang Vinjé, Jan J Clin Virol Short Communication BACKGROUND: Noroviruses are the major cause of acute gastroenteritis (AGE) in people of all ages globally. Standardized genotyping is key for outbreak investigations and surveillance networks. OBJECTIVE: Here we describe the validation of a one-step conventional RT-PCR assay for sequence-based dual typing of GI and GII noroviruses. This polymerase (P) and capsid (C) dual typing assay uses a combination of previously published oligonucleotide primers amplifying a genomic region spanning the 3'-end of ORF1 and 5'end of ORF2 resulting in a 579 bp product for GI and 570 bp product for GII viruses. RESULTS: The limit of detection of the assay ranged from 5 to 50 copies of viral RNA per reaction for GI and GII. To validate the assay, we tested 2,663 noroviruspositive stool samples from outbreaks and sporadic cases of AGE in Bangladesh, Guatemala, Peru, and USA collected between 2010–2019, of which 2,392 (90 %) were genotyped successfully. Most of the known genotypes infecting humans (GI (n = 9) and GII (n = 23)) and P types (GI (n = 15), GII, (n = 20)) could be detected. The remaining 270 samples had low viral load (Ct > 30) by real-time RT-PCR. A panel of 166 samples positive for other enteric viruses (rotavirus, astrovirus, sapovirus, adenovirus type 40/41) tested negative. CONCLUSION: The use of broadly reactive genotyping assays greatly strengthens exchange of standardized genotype data globally to monitor trends in genotype diversity which is important for both the development of vaccines and to measure their impact. Elsevier Science 2021-01 /pmc/articles/PMC7816162/ /pubmed/33260046 http://dx.doi.org/10.1016/j.jcv.2020.104689 Text en http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Short Communication Chhabra, Preeti Browne, Hannah Huynh, Thalia Diez-Valcarce, Marta Barclay, Leslie Kosek, Margaret N. Ahmed, Tahmeed Lopez, Maria Renee Pan, Chao-Yang Vinjé, Jan Single-step RT-PCR assay for dual genotyping of GI and GII norovirus strains |
title | Single-step RT-PCR assay for dual genotyping of GI and GII norovirus strains |
title_full | Single-step RT-PCR assay for dual genotyping of GI and GII norovirus strains |
title_fullStr | Single-step RT-PCR assay for dual genotyping of GI and GII norovirus strains |
title_full_unstemmed | Single-step RT-PCR assay for dual genotyping of GI and GII norovirus strains |
title_short | Single-step RT-PCR assay for dual genotyping of GI and GII norovirus strains |
title_sort | single-step rt-pcr assay for dual genotyping of gi and gii norovirus strains |
topic | Short Communication |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816162/ https://www.ncbi.nlm.nih.gov/pubmed/33260046 http://dx.doi.org/10.1016/j.jcv.2020.104689 |
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