TY - JOUR
T1 - Single-step RT-PCR assay for dual genotyping of GI and GII norovirus strains
AU - Chhabra, Preeti
AU - Browne, Hannah
AU - Huynh, Thalia
AU - Diez-Valcarce, Marta
AU - Barclay, Leslie
AU - Kosek, Margaret N.
AU - Ahmed, Tahmeed
AU - Lopez, Maria Renee
AU - Pan, Chao Yang
AU - Vinjé, Jan
N1 - Funding Information:
This study was partly supported by a grant from the intramural food safety program of CDC (PC, HB, LB, JV) and in part by the Bill & Melinda Gates Foundation ( OPP1066146 ) and 47075 to the Fogarty International Center (MNK) . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Publisher Copyright:
© 2020
PY - 2021/1
Y1 - 2021/1
N2 - 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.
AB - 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.
KW - Gastroenteritis
KW - Norovirus
KW - RT-PCR
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U2 - 10.1016/j.jcv.2020.104689
DO - 10.1016/j.jcv.2020.104689
M3 - Article
C2 - 33260046
AN - SCOPUS:85097444458
VL - 134
JO - Journal of Clinical Virology
JF - Journal of Clinical Virology
SN - 1386-6532
M1 - 104689
ER -